Modulation of gamma delta T cells to regulate airway hyperresponsiveness

ABSTRACT

Disclosed is a method for regulation of airway hyperresponsiveness by modulating the action of γδ T cells in a patient. Also disclosed are methods for identifying compounds that regulate airway hyperresponsiveness by modulating γδ T cell action.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/672,865, filed Sep. 28, 2000, which application claims priority under35 U.S.C. § 119(e) from U.S. Provisional Application Ser. No.60/157,231, filed Sep. 30, 1999, and entitled “Regulation of AirwayHyperresponsiveness by Modulation of y8 T Cells.” The entire disclosureof U.S. patent application Ser. No. 09/672,865 and U.S. ProvisionalApplication Ser. No. 60/157,231 is incorporated herein by reference.

GOVERNMENT RIGHTS

This invention was made in part with government support under NIH GrantHL-36577, NIH Grant AI-40611 and NIH Grant AI-01291, all awarded by theNational Institutes of Health. The government has certain rights to thisinvention.

FIELD OF THE INVENTION

The present invention generally relates to a method to regulate airwayhyperresponsiveness by modulating the action of γδ T cells in a patient.The present invention further relates to methods for identifyingcompounds that regulate airway hyperresponsiveness by modulating γδ Tcell action.

BACKGROUND OF THE INVENTION

Diseases involving inflammation are characterized by the influx ofcertain cell types and mediators, the presence of which can lead totissue damage and sometimes death. Diseases involving inflammation areparticularly harmful when they afflict the respiratory system, resultingin obstructed breathing, hypoxemia, hyperapnia and lung tissue damage.Obstructive diseases of the airways are characterized by airflowlimitation (i.e., airflow obstruction or narrowing) due to constrictionof airway smooth muscle, edema and hypersecretion of mucus leading toincreased work in breathing, dyspnea, hypoxemia and hypercapnia.

A variety of inflammatory agents can provoke airflow limitationincluding allergens, cold air, exercise, infections and air pollution.In particular, allergens and other agents in allergic or sensitizedmammals (i.e., antigens and haptens) cause the release of inflammatorymediators that recruit cells involved in inflammation. Such cellsinclude lymphocytes, eosinophils, mast cells, basophils, neutrophils,macrophages, monocytes, fibroblasts and platelets. Inflammation resultsin airway hyperresponsiveness (AHR). A variety of studies have linkedthe degree, severity and timing of the inflammatory process with thedegree of airway hyperresponsiveness. Thus, a common consequence ofinflammation is airway hyperresponsiveness.

Currently, therapy for treatment of inflammatory diseases involving AHR,such as moderate to severe asthma and chronic obstructive pulmonarydisease, predominantly involves the use of glucocorticosteroids andother anti-inflammatory agents. These agents, however, have thepotential of serious side effect, including, but not limited to,increased susceptibility to infection, liver toxicity, drug-induced lungdisease, and bone marrow suppression. Thus, such drugs are limited intheir clinical use for the treatment of lung diseases associated withairway hyperresponsiveness. The use of anti-inflammatory and symptomaticrelief reagents is a serious problem because of their side effects ortheir failure to attack the underlying cause of an inflammatoryresponse. There is a continuing requirement for less harmful and moreeffective reagents for treating inflammation. Thus, there remains a needfor processes using reagents with lower side effect profiles, lesstoxicity and more specificity for the underlying cause of AHR.

Airway hyperresponsiveness (AHR) is the result of complexpathophysiological changes in the airway. A variety of studies havelinked the degree, severity and timing of the inflammatory process withthe degree of airway hyperresponsiveness. However, the mechanismsleading to AHR are still poorly understood and can be attributed to bothimmune-dependent and immune-independent mechanisms. Essentially all ofthe T cell-mediated effects described so far are in the former category.However, T cells from hyperresponsive mice can increase baseline airwaytone in hyporesponsive mice after cell transfer. Because of theirconstitutive presence in the normal lung, γδ T cells have beeninvestigated with regard to their potential role in airway responses.

γδ T cells have been observed to proliferate and produce cytokines inmany diseases. In addition, studies in animal models have providedevidence that these cells contribute to host resistance againstinfections (Hiromatsu et al., 1992, J. Exp. Med. 175:49), and that theycan influence inflammation (Fu et al., 1994, J. Immunol. 153:3101),epithelial regeneration (Boismenu et al., 1994, Science 266:1253), andmucosal tolerance to antigens (Fujihashi et al., 1992, J. Exp. Med.175:695; McMenamin et al., 1994, supra). Investigators are stilldetermining what stimuli trigger γδ T cell reactivity, and to whatextent γδ T cell activating stimuli differ from those of αβ T cells andB lymphocytes. It is known that γδ T cells respond during bacterial andviral infections, although they have not been readily linked toantigen-specific adaptive immunity.

A number of studies have investigated the presence and role of γδ Tcells in diseases of the airways. Pawankar et al. noted the mucosalchanges at the site of allergic inflammation in patients with perennialallergic rhinitis and chronic infective rhinitis includes an oligoclonalexpansion and activation of Vγ1/Vδ⁺ T cells (Pawankar and Ra, 1996, J.Allergy Clin. Immunol. 98:S248-62). Molfino et al. showed that much ofthe γδ T cell population found in broncho alveolar lavage (BAL) fluid inhumans derives from clonally expanded T cells (Molfino et al., 1996,Clin. Exp. Iminunol. 104:144-153). Spinozzi et al., measuring γδ T cellsin the BAL fluid from patients with asthma, concluded thatallergen-specific, steroid-sensitive γδ T cells may be one of thecellular components involved in the airway inflammation thatcharacterizes allergic bronchial asthma (Spinozzi et al., 1996, Ann.Intern. Med. 124:223-227 and 1995, Mol. Med. 1:821-826).

Moreover, it has been noted that in patients with respiratory conditionsincluding Bordetella pertussin infection (whooping cough) and asthma,circulating γδ T cells are decreased. It has been suggested that thereason for this decrease is the dispatch of γδ T cells to the site ofinflammation in the lung. (Bertotto et al., 1997, Acta Paediatr.86:114-115; Schauer et al., 1991, Clin. Exp. Immunol. 86:440-443;Krejsek et al., 1998, Allergy 53;73-77).

Many of the studies directed to γδ T cells and airway diseases havedirectly suggested that γδ T cells are proinflammatory, promoting acuteairway sensitization, increases in cytokine levels suggested to beinvolved in allergic inflammation, regulation of allergic αβ T-cell andallergen specific B-cell responses, and/or allergen-induced eosinophiliaand IgE responses (e.g., McMenamin et al., 1994, Science 265:1869-1871;Zuany-Amorim et al., 1998, supra; Schramm et al., 2000, Am. J. Respir.Cell Mol. Biol. 22:218-225; Schramm et al., 1999, InternationalConference of the American Thoracic Society; vol. 159:A255 (AmericanJournal of Respiratory and Critical Care Medicine, San Diego, Calif.)).Some investigators, alternatively, have concluded that γδ T cells do notplay a significant role in airway allergic inflammation. For example,Chen et al. noted, similar to other investigators discussed above, thatallergic asthmatics have reduced γδ T cells in the peripheral blood.However, Chen et al. concluded that no significant correlation existedbetween the levels of γδ T cells and IgE present in the peripheral blood(Chen et al., 1996, Clin. Exp. Iminunol. 26:295-302). Although allergicasthmatics have reduced γδ T cells with reciprocally elevated eosinophilnumbers in the peripheral blood, Chen et al. asserted that this does notindicate that the reduction of γδ T cells correlates with thepredominance of eosinophilia or IgE levels in diseased populations.Jaffar et al. described a role for au, but not γδ, T cells inallergen-induced Th2 cytokine production from asthmatic bronchial tissue(Jaffar et al., 1999, J. Immunol. 163:6283-6291). Fajac et al., 1997,Eur. Resp. J 10:633-638 investigated the role of heat shock proteins andγδ T cells in patients with mild atopic asthma, and concluded thatneither heat shock proteins nor γδ T cells play an important role ininflammatory and immune responses in mild asthma.

Therefore, prior to the present invention, those of skill in the arteither considered γδ T cells to play an insignificant role, if any, indiseases of the airways, or believed that γδ T cells wereproinflammatory cells which contributed to the development of acuteairway hyperresponsiveness and other events associated withinflammation.

SUMMARY OF THE INVENTION

The present inventors have discovered that yb cells can regulate airwayfunction in an aid T cell-independent manner, identifying them asimportant cells in pulmonary homeostasis. This function of γδ T cellsdiffers from previously described immune-dependent mechanisms and mayreflect their interaction with innate systems ofhost defense.Specifically, in contrast to other studies that emphasized their role inthe modification of allergen-specific αβ T cell and B-cell responses,the present inventors have found that γδ T cells maintain normal airwayresponsiveness independently of αβ T cells.

One embodiment of the present invention relates to a method to reduceairway hyperresponsiveness in a mammal. The method includes the step ofincreasing γδ T cell action in a mammal that has, or is at risk ofdeveloping, a respiratory condition associated with airwayhyperresponsiveness. In one aspect, the step of increasing γδ T cellaction comprises increasing the number of γδ T cells in the lung tissueof the mammal. For example, the step of increasing can comprise removingγδ T cells from the mammal, inducing the γδ T cells to proliferate exvivo to increase the number of the γδ T cells, and returning the γδ Tcells to the lung tissue of the mammal. In another aspect, the step ofincreasing γδ T cell action comprises activating γδ T cells in themammal. Activating γδ T cells can be performed ex vivo or in vivo.

In one embodiment of the method, the step of increasing γδ T cell actioncomprises administering an agent to the mammal that activates γδ T cellsin the mammal. Such an agent can be any agent suitable for activating γδT cells. In one aspect, the agent is a protein comprising a BiP-bindingmotif, wherein the protein is administered in an amount effective toinduce proliferation of γδ T cells in the mammal. In another aspect, theagent is selected from the group consisting of a glycosylated proteinand a glycosylated peptide. In another aspect, the agent is selectedfrom the group consisting of polyGT and poly GAT (1:1:1). In yet anotherembodiment, the agent is selected from the group of: synthetic GC,synthetic AT and other oligonucleotides. In yet another aspect, theagent is a mycobacterial product. In another aspect, the agent is aListeria cell wall product. In another aspect, the agent is acardiolipin. In yet another aspect, the agent is tumor necrosis factor-α(TNF-α). In one aspect, the agent is an antibody that specifically bindsto a γδ T cell receptor and activates the γδ T cells. Preferably, theagent is an antibody that specifically binds to a γδ T cell receptor(TCR) from a γδ T cell subset that is particularly suitable forregulation of airway hyperresponsiveness. Such a TCR includes, but isnot limited to, a murine TCR comprising Vγ4 and a human TCR comprisingVγ1.

In one aspect of the method of the present invention, the agent istargeted to γδ T cells in the mammal. Preferably, the agent is targetedto γδ T cells in the lung tissue of the mammal. In one embodiment, theagent is targeted to γδ T cell subsets that are particularly suitablefor regulation of airway hyperresponsiveness, such γδ T cells having a Tcell receptor (TCR) selected from: a murine TCR comprising Vγ4 and ahuman TCR comprising Vγ1. In one aspect, the agent comprises: (a) anantibody that specifically binds to a molecule on the cell surface of γδT cells; and (b) a compound that activates the γδ T cells, wherein thecompound is linked to the antibody of (a). The compound can include, butis not limited to: a protein comprising a peptide having a BiP-bindingmotif, a glycosylated protein or peptide, polyGT, polyGAT (1:1:1),synthetic GC, synthetic AT, a mycobacterial product, a Listeria cellwall product, cardiolipin, TNF-α, and an antibody that specificallybinds to a γδ T cell receptor and activates the receptor.

In one aspect of the present method, the agent is administered to thelung tissue of the mammal. In a preferred embodiment, the agent isadministered by a route selected from the group consisting of inhaled,intratracheal and nasal routes. Preferably, the agent is administered tothe animal in an amount effective to reduce airway hyperresponsivenessin the animal as compared to prior to administration of the agent. Inone aspect, the agent is administered with a pharmaceutically acceptableexcipient.

Preferably, the method of the present invention increases γδ T cellaction within between about 1 hour and 6 days of an initial diagnosis ofairway hyperresponsiveness in the mammal. In another embodiment, the γδT cell action is increased within less than about 72 hours of an initialdiagnosis of airway hyperresponsiveness in the mammal. In anotherembodiment, the γδ T cell action is increased prior to development ofairway hyperresponsiveness in the mammal. Preferably, the step ofincreasing γδ T cell action decreases airway methacholine responsivenessin the mammal, and/or reduces airway hyperresponsiveness of the mammalsuch that the FEV₁ value of the mammal is improved by at least about 5%.It is also preferred that the step of increasing γδ T cell actionimproves the mammal's PC_(20methacholin)FEV₁ value such that thePC₂₀methacholineFEVI value obtained before the step of increasing γδ Tcell action when the mammal is provoked with a first concentration ofmethacholine is substantially the same as the PC_(20methacholin)FEV₁value obtained after increasing γδ T cell action when the mammal isprovoked with double the amount of the first concentration ofmethacholine. Preferably, the first concentration of methacholine isbetween about 0.01 mg/ml and about 8 mg/ml. The method of the presentinvention is suitable for treating airway hyperresponsiveness associatedwith any condition including, but not limited to, airwayhyperresponsiveness is associated with a disease selected from the groupconsisting of chronic obstructive disease of the airways and asthma.

Yet another embodiment of the present invention relates to a method toidentify a compound that reduces or prevents airway hyperresponsivenessassociated with inflammation. The method includes the steps of: (a)contacting a putative regulatory compound with a γδ T cell; (b)detecting whether the putative regulatory compound increases the actionof the γδ T cell; and, (c) administering the putative regulatorycompound to a non-human animal in which airway hyperresponsiveness canbe induced, and identifying animals in which airway hyperresponsivenessis reduced or prevented as compared to in the absence of the putativeregulatory compound. A putative regulatory compound that increases γδ Tcell action and that reduces or prevents airway hyperresponsiveness inthe non-human animal is indicated to be a compound for reducing orpreventing hyperresponsiveness. Preferably, step (b) of detecting isselected from the group consisting of measurement proliferation of theγδ T cell, measurement of cytokine production by the γδ T cell,measurement of calcium mobilization in the γδ T cell, measurement ofcytokine receptor expression by the γδ T cell, measurement of.CD69upregulation by the γδ T cell, measurement of upregulation, of CD44 bythe γδ T cell, and measurement of cytoskeletal reorganization by the γδT cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a line graph showing changes in airway resistance (R_(L)) innormal C57BL/6 mice after systemic airway sensitization, aerosol onlyairway sensitization, and no treatment.

FIG. 1B is a line graph showing changes in airway resistance (R_(L)) inTCR-δ^(−\−) mice, TCR-β^(−\−) mice and normal C57BL/6 after systemicairway sensitization.

FIG. 1C is a line graph showing changes in airway resistance (R_(L)) inTCR-δ-depleted or sham-depleted BALB/c mice after systemic airwaysensitization.

FIG. 1D is a bar graph showing BAL fluid cell composition for totalcells, eosinophils and macrophages in C57BL/6 mice, TCR-δ^(−\−) mice andTCR-β^(−\−) mice after systemic airway sensitization.

FIG. 2A is a line graph showing changes in airway resistance (R_(L)) inC57BL/6 mice, TCR-δ^(−\−) mice and TCR-β^(−\−) mice after aerosol onlyairway sensitization.

FIG. 2B is a line graph showing changes in dynamic compliance (C_(dyn))in C57BL/6 mice, TCR-δ^(−\−) mice and TCR-β^(−/−) mice after aerosolonly airway sensitization.

FIG. 2C is a line graph showing changes in airway resistance (R_(L)) insham-depleted C57BL/6 mice and γδ T cell-depleted C57BL/6 mice afteraerosol only airway sensitization.

FIG. 2D is a line graph showing changes in dynamic compliance (C_(dyn))in sham-depleted C57BL/6 mice and γδ T cell-depleted C57BL/6 mice afteraerosol only airway sensitization.

FIG. 2E is a line graph showing changes in airway resistance (RL)sham-depleted BALB/c mice and γδ T cell-depleted BALB/c mice afteraerosol only airway sensitization.

FIG. 2F is a line graph showing changes in dynamic compliance (C_(dyn))sham-depleted BALB/c mice and γδ T cell-depleted BALB/c mice afteraerosol only airway sensitization.

FIG. 2G is a bar graph showing BAL fluid cell composition for totalcells, eosinophils and macrophages in C57BL/6 mice, TCR-δ^(−\−) mice andTCR-β^(−\−) mice after aerosol only airway sensitization.

FIG. 2H is a bar graph showing BAL fluid cell composition for totalcells, eosinophils and macrophages in sham-depleted C57BL/6 mice and γδT cell-depleted C57BL/6 mice after aerosol only airway sensitization.

FIG. 2I is a bar graph showing BAL fluid cell composition for totalcells, eosinophils and macrophages in sham-depleted BALB/c mice and γδ Tcell-depleted BALB/c mice after aerosol only airway sensitization.

FIG. 3A is a line graph showing changes in airway resistance (RL) insham-depleted TCR-β^(b−\−) mice and γδ-depleted TCR-β^(−\−) mice afteraerosol only airway sensitization.

FIG. 3B is a line graph showing changes in dynamic compliance (Cdyn) insham-depleted TCR-β^(−\−) mice and y8-depleted TCR-β^(−\−) mice afteraerosol only airway sensitization.

FIG. 3C is a line graph showing changes in airway resistance (R_(L)) insham-depleted TCR-β^(−\−) mice and αβ-depleted TCR-β^(−\−) mice afteraerosol onlyairway sensitization.

FIG. 3D is a line graph showing changes in dynamic compliance (C_(dyn))in sham-depleted TCR-β^(−\−) mice and αβ-depleted TCR-β^(−\−) mice afteraerosol only airway sensitization.

FIG. 3E is a bar graph showing BAL fluid cell composition for totalcells, eosinophils and macrophages in sham-depleted TCR-β^(−\−) mice andy8-depleted TCR-β^(−\−) mice after aerosol only airway sensitization.

FIG. 3F is a bar graph showing BAL fluid cell composition for totalcells, eosinophils and macrophages in sham-depleted TCR-β^(−\−) mice andap-depleted TCR-β^(−\−) mice after aerosol only airway sensitization.

FIG. 4A is a graph showing serum levels of OVA-specific IgG₁ in C57BL/6mice, TCRβ_(−\−) mice, and TCR-δ_(−\−) mice after aerosol only andsystemic airway sensitization.

FIG. 4B is a graph showing serum levels of OVA-specific IgE n C57BL/6mice, TCR-β_(−\−) mice, and TCR-δ_(−\−) mice after aerosol only andsystemic airway sensitization.

FIG. 4C is a graphshowing serum levels of OVA-specific IgG_(2a) inC57BL/6 mice, TCR-β_(−\−) mice, and TCR-δ_(−\−) mice after aerosol onlyand systemic airway sensitization.

FIG. 5A is a bar graph showing BAL fluid IL-5 levels in C57BL/6 mice,TCR-β_(−\−) mice, and TCR-δ_(−\−) mice after aerosol only and systemicairway sensitization.

FIG. 5B is a bar graph showing BAL fluid IL-4 levels in C57BL/6 mice,TCR-β_(−\−) mice, and TCR-δ_(−\−) mice after aerosol only and systemicairway sensitization.

FIG. 5C is a bar graph showing BAL fluid IFN-γ levels in C57BL/6 mice,TCR-β_(−\−) mice, and TCR-δ_(−\−) mice after aerosol only and systemicairway sensitization.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to a method to reduce or preventairway hyperresponsiveness (AHR) in an animal that has, or is at risk ofdeveloping, airway hyperresponsiveness, by increasing the action of γδ Tcells (i.e., γδ T lymphocytes) in the animal. In the method of thepresent invention, the animal has, or is at risk of developing, airwayhyperresponsiveness associated with inflammation. For example, airwayhyperresponsiveness is commonly associated with allergic inflammationand/or viral-induced inflammation. Airway hyperresponsiveness associatedwith allergic inflammation can occur in a patient that has, or is atrisk of developing, a condition including, but not limited to, anychronic obstructive disease of the airways. Such conditions include, butare not limited to: asthma, chronic obstructive pulmonary disease,allergic bronchopulmonary aspergillosis, hypersensitivity pneumonia,eosinophilic pneumonia, emphysema, bronchitis, allergic bronchitisbronchiectasis, cystic fibrosis, tuberculosis, hypersensitivitypneumonitis, occupational asthma, sarcoid, reactive airway diseasesyndrome, interstitial lung disease, hyper-eosinophilic syndrome,rhinitis, sinusitis, exercise-induced asthma, pollution-induced asthmaand parasitic lung disease. Airway hyperresponsiveness associated withviral-induced inflammation can occur in a patient that has, or is atrisk of developing, an infection by a virus including, but not limitedto, respiratory syncytial virus (RSV), parainfluenza virus (PIV),rhinovirus (RV) and adenovirus.

The present invention is based on the present inventors' discovery thatγδ T cells maintain normal airway responsiveness independently of αβ Tcells and that the increased action of γδ T cells in patient's thathave, or are at risk of developing, airway hyperresponsiveness will havea beneficial effect. To define the role of γδ T cells in controlling thedevelopment of AHR, the present inventors used an established mousemodel of eosinophilic airway inflammation and allergen-drivenalterations in airway function. The results of this researchdemonstrated a previously unknown, γδ T cell-dependent mechanism in theregulation of airway responsiveness which is independent of αβ T cellsand their allergen-specific responses. Furthermore, the presentinventors' have found no evidence to indicate that antibodies areinvolved in this regulatory mechanism or that changes in cytokine levelspreviously suggested to be involved in models allergic inflammation areinvolved (McMenamin et al., 1994, Science 265:186-1871; Zuany-Amorim etal., 1998, supra). The present inventors' discovery was surprising,because the results differ from earlier reports, which have emphasizedthe role of γδ T cells in regulating allergic αβ T-cell and allergenspecific B-cell responses, or their role in promoting allergen-inducedeosinophilia and IgE responses (McMenamin et al., 1994, supra;Zuany-Amorim et al., 1998, supra; Schramm et al., 1999, InternationalConference of the American Thoracic Society; vol. 159:A255 (AmericanJournal of Respiratory and Critical Care Medicine, San Diego, Calif.)).The mechanism of γδ T cell-dependent regulation of airway responsesdescribed herein is therefore not restricted to allergic inflammation.

One embodiment of the present invention relates to a method to reduce orprevent airway hyperresponsiveness in an animal. This method includes astep of increasing γδ T cell action in a mammal that has, or is at riskof developing, a respiratory condition associated with airwayhyperresponsiveness. According to the present invention, “airwayhyperresponsiveness” or “AHR” refers to an abnormality of the airwaysthat allows them to narrow too easily and/or too much in response to astimulus capable of inducing airflow limitation. AHR can be a functionalalteration of the respiratory system caused by inflammation or airwayremodeling (e.g., such as by collagen deposition). Airflow limitationrefers to narrowing of airways that can be irreversible or reversible.Airflow limitation and/or airway hyperresponsiveness can be caused bycollagen deposition, bronchospasm, airway smooth muscle hypertrophy,airway smooth muscle contraction, mucous secretion, cellular deposits,epithelial destruction, alteration to epithelial permeability,alterations to smooth muscle function or sensitivity, abnormalities ofthe lung parenchyma and/or infiltrative diseases in and around theairvays. Many of these causative factors can be associated withinflammation. The present invention is directed to any airwayhyperresponsiveness, including airway hyperresponsiveness that isassociated with inflammation of the airways, eosinophilia andinflammatory cytokine production. Methods of measuring and monitoringAHR are discussed in detail below.

As used herein, to reduce airway hyperresponsiveness refers to anymeasurable reduction in airway hyperresponsiveness and/or any reductionof the occurrence or frequency with which airway hypertesponsivenessoccurs in a patient. A reduction in AHR can be measured using any of theabove-described techniques or any other suitable method known in theart. Preferably, airway hyperresponsiveness, or the potential therefore,is reduced, optimally, to an extent that the animal no longer suffersdiscomfort and/or altered function resulting from or associated withairway hyperresponsiveness. To prevent airway hyperresponsiveness refersto preventing or stopping the induction of airway hyperresponsivenessbefore biological characteristics of airway hyperresponsiveness asdiscussed above can be substantially detected or measured in a patient.

AHR can be measured by a stress test that comprises measuring ananimal's respiratory system function in response to a provoking agent(i.e., stimulus). AHR can be measured as a change in respiratoryfunction from baseline plotted against the dose of a provoking agent (aprocedure for such measurement and a mammal model useful therefore aredescribed in detail below in the Examples). Respiratory function can bemeasured by, for example, spirometry, plethysmograph, peak flows,symptom scores, physical signs (i.e., respiratory rate), wheezing,exercise tolerance, use of rescue medication (i.e., bronchodialators)and blood gases. In humans, spirometry can be used to gauge the changein respiratory function in conjunction with a provoking agent, such asmethacholine or histamine. In humans, spirometry is performed by askinga person to take a deep breath and blow, as long, as hard and as fast aspossible into a gauge that measures airflow and volume. The volume ofair expired in the first second is known as forced expiratory volume(FEV₁) and the total amount of air expired is known as the forced vitalcapacity (FVC). In humans, normal predicted FEV₁ and FVC are availableand standardized according to weight, height, sex and race. Anindividual free of disease has an FEV, and a FVC of at least about 80%of normal predicted values for a particular person and a ratio ofFEV₁/FVC of at least about 80%. Values are determined before (i.e,representing a mammal's resting state) and after (i.e., representing amammal's higher lung resistance state) inhalation of the provokingagent. The position of the resulting curve indicates the sensitivity ofthe airways to the provoking agent.

The effect of increasing doses or concentrations of the provoking agenton lung function is determined by measuring the forced expired volume in1 second (FEV₁) and FEV₁ over forced vital capacity (FEV₁/FVC ratio) ofthe mammal challenged with the provoking agent. In humans, the dose orconcentration of a provoking agent (i.e., methacholine or histamine)that causes a 20% fall in FEV₁ (PD₂₀FEV₁) is indicative of the degree ofAHR. FEV₁ and FVC values can be measured using methods known to those ofskill in the art.

Pulmonary function measurements of airway resistance (R_(L)) and dynamiccompliance (C_(dyn) or C_(L)) and hyperresponsiveness can be determinedby measuring transpulmonary pressure as the pressure difference betweenthe airway opening and the body plethysmograph. Volume is the calibratedpressure change in the body plethysmograph and flow is the digitaldifferentiation of the volume signal. Resistance (R_(L)) and compliance(C_(L)) are obtained using methods known to those of skill in the art(e.g., such as by using a recursive least squares solution of theequation of motion). The measurement of lung resistance (R_(L)) anddynamic compliance (C_(L)) are described in detail in the Examples. Itshould be noted that measuring the airway resistance (R_(L)) value in anon-human mammal (e.g., a mouse) can be used to diagnose airflowobstruction similar to measuring the FEV₁ and/or FEV₁/FVC ratio in ahuman.

A variety of provoking agents are useful for measuring AHR values.Suitable provoking agents include direct and indirect stimuli. Preferredprovoking agents include, for example, an allergen, methacholine, ahistamine, a leukotriene, saline, hyperventilation, exercise, sulfurdioxide, adenosine, propranolol, cold air, an antigen, bradykinin,acetylcholine, a prostaglandin, ozone, environmental air pollutants andmixtures thereof. Preferably, Mch is used as a provoking agent.Preferred concentrations of Mch to use in a concentration-response curveare between about 0.001 and about 100 milligram per milliliter (mg/ml).More preferred concentrations of Mch to use in a concentration-responsecurve are between about 0.01 and about 50 mg/ml. Even more preferredconcentrations of Mch to use in a concentration-response curve arebetween about 0.02 and about 25 mg/ml. When Mch is used as a provokingagent, the degree of AHR is defined by the provocative concentration ofMch needed to cause a 20% drop of the FEV₁ of a mammal(PC_(20methacholine)FEV₁). For example, in humans and using standardprotocols in the art, a normal person typically has aPC_(20methacholine)FEV₁>8 mg/ml of Mch. Thus, in humans, AHR is definedas PC_(20methacholine)FEV₁<8 mg/ml of Mch.

According to the present invention, respiratory function can also beevaluated with a variety of static tests that comprise measuring ananimal's respiratory system function in the absence of a provokingagent. Examples of static tests include, for example, spirometry,plethysmographically, peak flows, symptom scores, physical signs (i.e.,respiratory rate), wheezing, exercise tolerance, use of rescuemedication (i.e., bronchodialators) and blood gases. Evaluatingpulmonary function in static tests can be performed by measuring, forexample, Total Lung Capacity (TLC), Thoracic Gas Volume (TgV),Functional Residual Capacity (FRC), Residual Volume (RV) and SpecificConductance (SGL) for lung volumes, Diffusing Capacity of the Lung forCarbon Monoxide (DLCO), arterial blood gases, including pH, P_(O2) andP_(CO2) for gas exchange. Both FEV₁ and FEV₁/FVC can be used to measureairflow limitation. If spirometry is used in humans, the FEV₁ of anindividual can be compared to the FEV₁ of predicted values. PredictedFEV₁ values are available for standard normograms based on the animal'sage, sex, weight, height and race. A normal animal typically has an FEV₁at least about 80% of the predicted FEV₁ for the animal. Airflowlimitation results in a FEV₁ or FVC of less than 80% of predictedvalues. An alternative method to measure airflow limitation is based onthe ratio of FEV₁ and FVC (FEV₁/FVC). Disease free individuals aredefined as having a FEV₁/FVC ratio of at least about 80%. Airflowobstruction causes the ratio of FEV₁/FVC to fall to less than 80% ofpredicted values. Thus, an animal having airflow limitation is definedby an FEV₁/FVC less than about 80%.

In one embodiment, the method of the present invention decreasesmethacholine responsiveness in the animal. Preferably, the method of thepresent invention results in an improvement in a mammal'sPC_(20methacholine)FEV₁ value such that the PC_(20methacholine)FEV₁value obtained before use of the present method when the mammal isprovoked with a first concentration of methacholine is the same as thePC_(20methacholine)FEV₁ value obtained after use of the present methodwhen the mammal is provoked with double the amount of the firstconcentration of methacholine. Preferably, the method of the presentinvention results in an improvement inma mammal'sPC_(20methacholine)FEV₁ value such that the PC20mcthacholineFEV₁ valueobtained before the use of the present method when the animal isprovoked with between about 0.01 mg/ml to about 8 mg/ml of methacholineis the same as the PC_(20methacholine)FEV, value obtained after the useof the present method when the animal is provoked with between about0.02 mg/ml to about 16 mg/ml of methacholine.

In another embodiment, the method of the present invention improves ananimal's FEV₁ by at least about 5%, and more preferably by between about6% and about 100%, more preferably by between about 7% and about 100%,and even more preferably by between about 8% and about 100% of themammal's predicted FEV₁. In another embodiment, the method of thepresent invention improves an animal's FEV₁ by at least about 5%, andpreferably, at least about 10%, and even more preferably, at least about25%, and even more preferably, at least about 50%, and even morepreferably, at least about 75%.

In yet another embodiment, the method of the present invention resultsin an increase in the PC_(20methacholine)FEV₁ of an animal by about onedoubling concentration towards the PC_(20methacholine)FEV₁ of a normalanimal. A normal animal refers to an animal known not to suffer from orbe susceptible to abnormal AHR. A patient, or test animal refers to ananimal suspected of suffering from or being susceptible to abnormal AHR.

Therefore, an animal that has airway hyperresponsiveness is an animal inwhich airway hyperresponsiveness is measured or detected, such as byusing one of the above methods for measuring airway hyperresponsiveness.To be associated with inflammation, the airway hyperresponsiveness isapparently or obviously, directly or indirectly associated with (e.g.,caused by, a symptom of, indicative of, concurrent with) an inflammatorycondition or disease (i.e., a condition or disease characterized byinflammation). Typically, such an inflammatory condition or disease isat least partially characterized by inflammation of pulmonary tissues.Such conditions or diseases are discussed above. An animal that is atrisk of developing airway hyperresponsiveness can be an animal that hasa condition or disease which is likely to be associated with at least apotential for airway hyperresponsiveness, but does not yet display ameasurable or detectable characteristic or symptom of airwayhyperresponsiveness. An animal that is at risk of developing airwayhyperresponsiveness also includes an animal that is identified as beingpredisposed to or susceptible to such a condition or disease.

Inflammation is typically characterized by the release of inflammatorymediators (e.g., cytokines or chemokines) which recruit cells involvedin inflammation to a tissue. For example, a condition or diseaseassociated with allergic inflammation is a condition or disease in whichthe elicitation of one type of immune response (e.g., a Th2-type immuneresponse) against a sensitizing agent, such as an allergen, can resultin the release of inflammatory mediators that recruit cells involved ininflammation in a mammal, the presence of which can lead to tissuedamage and sometimes death. Airway hyperresponsiveness associated withallergic inflammation can occur in a patient that has, or is at risk ofdeveloping, any chronic obstructive disease of the airways, including,but not limited to, asthma, chronic obstructive pulmonary disease,allergic bronchopulmonary asp ergillosis, hypersensitivity pneumonia,eosinophilic pneumonia, emphysema, bronchitis, allergic bronchitisbronchiectasis, cystic fibrosis, tuberculosis, hypersensitivitypneumonitis, occupational asthma, sarcoid, reactive airway diseasesyndrome, interstitial lung disease, hyper-eosinophilic syndrome,rhinitis, sinusitis, exercise-induced asthma, pollution-induced asthmaand parasitic lung disease. Preferred conditions to treat using themethod of the present invention include asthma, chronic obstructivedisease of the airways, occupational asthma, exercise-induced asthma,pollution-induced asthma and reactive airway disease syndrome, withchronic obstructive disease of the airways and asthma being particularlypreferred for treatment. Viral-induced inflammation typically involvesthe elicitation of another type of immune response (e.g., a Th1-typeimmune response) against viral antigens, resulting in production ofinflammatory mediators the recruit cells involved in inflammation in aan animal, the presence of which can also lead to tissue damage. Aiiwayhyperresponsiveness associated with viral-induced inflammation can occurin a patient that has, or is at risk of developing, an infection by avirus including, but not limited to, respiratory syncytial virus (RSV),parainflulenza virus (PIV), rhinovirus (RV) and adenovirus.

In order to reduce airway hypeiresponsiveness according to the method ofthe present invention, the action of γδ T cells is increased in ananimal that has, or is at risk of developing AHR, including AHRassociated with inflammation. A “γδ T cell” is a distinct lineage of Tlymphocytes found in mammalian species and birds that expresses aparticular antigen receptor (i.e., T cell receptor or TCR) that includesa γ chain and a δ chain. The γ and δ chains are distinguished from the αand β chains that make up the TCR of the perhaps more commonlyreferenced T cells known as “αβ T cells”. The γδ heterodimer of the γδ Tcells is expressed on the surface of the T cell and, like the αβheterodimer of αβ T cells, is associated with the CD3 complex on thecell surface. The γ and δ chains of the γδ T cell receptor should not beconfused with the γ and δ chains of the CD3 complex. According to thepresent invention, the terms “T lymphocyte” and “T cell” can be usedinterchangeably herein.

According to the present invention, to increase the action of γδ T cellsin an animal refers to any treatment or manipulation of the animal, orspecifically, of γδ T cells, which results in a detectable (e.g.,measurable) increase (i.e., enhancement, upregulation, induction,stimulation) in the number, activation, biological activity and/orsurvivability of the γδ T cells. Therefore, increasing the action of γδT cells according to the present invention can be accomplished byincreasing the number of γδ T cells in an animal (i.e., by causing thecells to proliferate/expand or by recruiting additional γδ T cells to asite), by increasing the activation of γδ T cells in an animal, byincreasing biological activity of γδ T cells (e.g., effector functionsor other activities of the cell) in an animal and/or by increasing theability of γδ T cells to survive (i.e., resist apoptosis) in an animal.According to the present invention, to increase the action of γδ T cellsin an animal further refers to a step of directly acting on γδ T cellsin the animal. In other words, the method of increasing the action of γδT cells directly expands, recruits, activates, or enhances survival ofγδ T cells, even though other cell types might be affected by themethod, but such step is not intended to be merely a downstream resultof a direct action on another cell type. Preferably, and particularlywhen the method is performed in vivo, the step of increasing the actionof γδ T cells does not substantially directly affect (i.e., act on)other cells, such as αβ T cells, B cells, macrophages, or monocytes. Inthis case, selective or targeted methods for increasing γδ T cells arepreferred. The increased action of γδ T cells can subsequently affectother cells, however, such as alveolar macrophages, airway epithelialand airway smooth muscle cells (i.e., increased numbers and/or activityof γδ T cells can influence the activity of other cells). It will beappreciated by those of skill in the art that when the step ofincreasing the action of γδ T cells is performed ex vivo or in vitro,the step of increasing γδ T cell action does not necessarily have to beselective for or targeted to γδ T cells, but preferably, γδ T cells aresubsequently isolated and/or preferentially returned to the animal.Therefore, in the preferred embodiment, the method of the presentinvention is intended to be selective for or specifically targeted to γδT cell activity, and in one embodiment, excludes methods whichindiscriminately activate other immune system cells and/or other celltypes, as well as methods which modulate γδ T cell activity as adownstream result of a direct action on another cell type.

More specifically, in one embodiment, an increase in γδ T cell action isdefined herein as any detectable increase in the number of γδ T cells ina population (clonal or non-clonal) of γδ T cells. According to thepresent invention, an increase in the number of γδ T cells at a givensite can be accomplished by: (1) causing a given population of γδ Tcells to proliferate and expand; (2) inducing recruitment of additionalγδ T cells to a given site, such that the total number of γδ T cellsincreases; and/or (3) adding additional γδ T cells to a population of Tcells (e.g., T cell transfer). An increase in the number of γδ T cellsis typically evaluated by measuring proliferation of γδ T cells, forexample, by using a standard T cell proliferation assay (e.g., uptake of[³H]-thymidine). T cell proliferation assays, including those using γδ Tcells, are well known in the art, and are described, for example, inseveral publications by certain of the present inventors (e.g., Born etal., 1990, Science 249:67; O'Brien et al., 1992, Proc. Natl. Acad. Sci.USA 89:4348; Lahn et al., 1998, J. Immunol. 160:5221; Cady et al., 2000,J. Immunol. 165:1790; all incorporated herein by reference in theirentireties). Other methods for determining an increase in the number ofγδ T cells can be evaluated by detecting or measuring the expressionlevel, and/or the distribution of γ-chain usage and/or δ chain usage inthe receptors of a population of γε T cells and determining whetherthere is a change in the expression level and/or distribution of one ormore γδ T cell receptor types in the population. Such assays, includingboth molecular and flow cytometric methods, and the reagents (e.g.,antibodies, hybridization probes and PCR primers specific for various γδTCR chains) for performing such assays, are known in the art (e.g.,O'Brien et al., 1992, supra; Lahn et al., 1998, supra; Cady et al.,2000, supra).

In another embodiment, an increase in γδ T cell action is any detectableincrease in the activation state and/or biological activity of γε Tcells in an animal. As used herein, activation, or responsiveness, of aγδ T cell refers to the ability of a γδ T cell to be activated by (e.g.,respond to) antigenic and/or mitogenic stimuli which results ininduction of γδ T cell activation signal transduction pathways andactivation events. The biological activity of a γδ T cell refers to anyfunction(s) exhibited or performed by a naturally occurring γδ T cell asmeasured or observed in vivo (i.e., in the natural physiologicalenvironment of the cell) or in vitro (i.e., under laboratoryconditions). As used herein, antigenic stimulation is stimulation of aγδ T cell by binding of the γδ T cell receptor to an antigen that isspecifically recognized by the γδ T cell in the context of appropriatecostimulatory signals necessary to achieve γδ T cell activation.Mitogenic stimulation is defined herein as any non-antigen stimulationof T cell activation, including by mitogens (lipopolysaccharides (LPS),phorbol esters, ionomycin) and antibodies (anti-TCR, anti-CD3, includingdivalent and tetravalent antibodies). Both antigenic stimulation and theforms of mitogenic stimulation which act at the level of the T cellreceptor (i.e., anti-TcR/CD3) result in T cell receptor-mediatedactivation, whereas LPS/phorbol ester/ionomycin mitogenic stimulationbypasses the T cell receptor and therefore, do not induce T cellreceptor-mediated activation, but nonetheless, can induce at least someof the downstream events of T cell activation.

Therefore, events associated with T cell activation or biologicalactivity include, but are not limited to, T cell proliferation, cytokineproduction (e.g., interleukin-2 (IL-2), IL-4, IL-5, IL-10, interferon-γ(IFN-γ), tumor necrosis factor-α (TNF-α)), upregulation of cytokinereceptors (e.g., IL-2 receptor, TNF-α receptor), calcium mobilization,upregulation of cell surface molecules associated with T cell activation(e.g., CD44, CD69), upregulation of expression and activity of signaltransduction proteins associated with T cell activation, chemokineproduction, altered T cell migration, accumulation of T cells atspecific tissue sites and/or cytoskeletal reorganization. The ability ofa T lymphocyte to respond, or become activated, by an antigenic ormitogenic stimulus can be measured by any suitable method of measuring Tcell activation. Such methods are well known to those of skill in theart. For example, after a T cell has been stimulated with an antigenmcor mitogenic stimulus, characteristics of T cell activation can bedetermined by a method including, but not limited to: measuring cytokineproduction by the T cell (e.g., by immunoassay or biological assay);measuring intracellular and/or extracellular calcium mobilization (e.g.,by calcium mobilization assays); measuring T cell proliferation (e.g.,by proliferation assays such as radioisotope incorporation); measuringupregulation of cytokine receptors on the T cell surface, includingIL-2R (e.g., by flow cytometry, immunofluorescence assays, immunoblots,RNA assays); measuring upregulation of other receptors associated with Tcell activation on the T cell surface (e.g., by flow cytometry,immunofluorescence assays, immunoblots, RNA assays); measuringreorganization of the cytoskeleton (e.g., by immunofluorescence assays,immunoprecipitation, immunoblots); measuring upregulation of expressionand activity of signal transduction proteins associated with T cellactivation (e.g., by kinase assays, phosphorylation assays, immunoblots,RNA assays); and, measuring specific effector functions of the T cell(e.g., by proliferation assays). Methods for performing each of thesemeasurements are well known to those of ordinary skill in the art, manyare described in detail or by reference to publications herein, and allsuch methods are encompassed by the present invention.

In another embodiment, an increase in γδ T cell action results in anincrease in the survival of the γδ T cell (i.e., prevention orinhibition of apoptosis). According to the present invention, thepresent method preferably results in a measurable maintenance of γδ Tlymphocyte survival (e.g., less than about 50%, and more preferably,less than about 25%, and more preferably, less than about 10%, and evenmore preferably, less than about 5% loss in blood γδ T lymphocyte numberafter employing the present method as compared to in the absence of thepresent method). T lymphocyte survival can be determined by measuringany of the parameters described above for measuring T cellproliferation/numbers or activation (as an indicator of responsive Tcells), or by any suitable means of measuring T cell apoptosis (i.e., areduction in γδ T cell apoptosis is indicative of enhanced γδ T cellsurvival). Methods of measuring apoptosis in a T cell include, but arenot limited to: determining the extent of a morphological change in acell; determining the extent of DNA cleavage by gel electrophoresis,cell cycle analysis, or in situ tailing or nick translation; assessingmembrane permeability by using dyes that bind RNA or DNA or Annexin V.Such methods are well known in the art.

According to the present invention, the method for regulating airwayhyperresponsiveness can be directed to any γδ T cell, wherein anincrease in the action of such γδ T cell results in a decrease in airwayhyperresponsiveness. Preferred γδ T cells to activate and/or expand(i.e., proliferate, increase the numbers) are γδ T cells in the lungtissue of an animal. Such γδ T cells include γδ T cells that normallyreside in the lung tissue, as well as γδ T cells that are recruited intothe lung upon development of a condition associated with airwayhyperresponsiveness and/or upon stimulation of γδ T cells that normallyreside in the lung tissue. Preferably, the present method includes astep of increasing γδ T cell action in γδ T cells that normally residein the lung tissue.

In another preferred embodiment, the method for regulation of airwayhyperresponsiveness of the present invention is directed to γδ T cellsthat are identified as being particularly useful for regulating AHR inan animal, wherein increased action of γδ T cells that do not regulateAHR, or which are proinflammatory (i.e., contribute to AHR), is avoided.In one aspect, a preferred γδ T cell for which increasing the action isbelieved to be particularly effective for reducing AHR has a T cellreceptor (TCR) that comprises a Vγ4 chain (i.e., the variable (V) regionof the γ chain is has a particular sequence which is known in the art asVγ4, following the nomenclature of Tonegawa et al., for example), or thehuman equivalent thereof, which is believed to include Vδ1 T cells(i.e., Vγ4 is the murine cell subset). Preferably, γδ T cells havingTCRs with Vγ4 chains, or the human equivalent (e.g., Vδ1), are targetedby the method of the present method. This subset of γδ T cells can betargeted, for example, by using a targeting moiety that selectivelyrecognizes the Vγ4 chain of the TCR (e.g., an antibody that selectivelybinds to Vγ4) (or the Vb 1 chain of the TCR in humans, for example), orby removing cells from the lung tissue (or other tissues) and isolatingγδ T cells expressing Vγ4 (or human equivalent) ex vivo.

In yet another preferred embodiment, γδ T cells that are CD8⁺ (i.e.,which express CD8) are preferred targets for the method of the presentinvention. Even more preferably, γδ T cells which express an αβheterodimer of CD8 are preferably selectively targeted for activationand/or expansion according to the present method. CD8 is a costimulatorymolecule expressed by subsets of both αβ T lymphocytes and γδ Tlymphocytes. The CD8 molecule comprises two chains which can occur inthe form of either a dimer of CD8α chains (i.e., a CD8α homodimer) or adimer of a CD8α chain and a CD8β chain (i.e., a CD8 at heterodimer). Inαβ T cells, the CD8 molecule is typically expressed as a CD8 αβheterodimer. In contrast, in γδ T cells, the CD8 molecule is typicallyexpressed as a CD8 α homodimer. However, the present inventors havefound that a subset of γδ T cells in the lung expresses CD8 as a CD8 αβheterodimer. Moreover, the present inventors have found that a subset ofVγ4⁺ T cells in the murine lung express the CD8 αβ heterodimer. Withoutbeing bound by theory, the present inventors believe that γδ T cellsexpressing a CD8 αβ heterodimer, and particularly γδ T cells expressingVγ4 (or the human equivalent such as V81) and a CD8 αβ heterodimer, areparticularly suitable targets for the method of the present inventionand are likely to be at least one primary regulatory γδ T cell subsetthat contributes to the reduction of AHR in vivo.

In another embodiment of the present invention, the method forregulation of ainvay hyperresponsiveness of the present invention isfurther directed to the inhibition of γδ T cells that are identified asbeing particularly enhancing of AHR in an animal, wherein decreasedaction of these γδ T cells that do not control AHR, and/or which areproinflammatory (i.e., contribute to AHR), is the goal. Without beingbound by theory, the present inventors believe that certain subsets ofγδ T cells appear to be enhancing of AHR and therefore, their targetedablation would be beneficial in the treatment of AER. In particular, thepresent inventors have discovered that γδ T cells bearing a T cellreceptor comprising a Vγ1 chain for murine cells (or the humanequivalent, such as Vγ9NV52 expressing cells), which appear later thanthe regulatory Vγ1 subset discussed above, may enhance AHR. Morespecifically, it is believed that the CD4⁺ Vγ1⁺ γδ T cell subset (or thehuman equivalent thereof) is a particularly desirable target forinhibition by the method of the present invention. Therefore, in oneembodiment of the present invention, either alone or in combination withthe stimulation of Vγ4+T cells (or the human equivalent thereof)according to the present method, Vγ1⁺ T cells (or the human equivalentthereof) are inhibited. Methods for inhibition will be clear to those ofskill in the art and include, but are not limited to targeteddestruction of Vγ1⁺ T cells (or the human equivalent thereof) (e.g., byneutralizing antibodies, induced apoptosis), blocking of such TCRs byblocking antibodies (i.e., that do not stimulate the T cell), anti-sensetherapy, and other such methods.

It is to be understood, however, that it is not necessary to selectivelytarget a particular subset of γδ T cells to reduce AHR in an animal, asmethods of increasing the action of γδ T cells which do not selectivelytarget a particular subset are also effective for reducing AHR. Forexample, in one embodiment, γδ T cell activation that is effective forreducing AHR can be selectively targeted or enhanced by increasing γδ Tcell action relatively early after airw % ay hyperresponsiveness (orinitial antigen sensitization leading to AHR) is induced. Without beingbound by theory, the present inventors believe that the γδ T cellresponses which are effective to downregulate AHR are most effectivewithin between about 1 hour to about 6 days after AHR is induced, andmost preferably, within less than about 72 hours after AHR is induced.As discussed above, it is further believed that γδ T cells which mayenhance AIHR appear later in the response, and could be avoided by earlytargeting, or actively ablated by later targeted delivery of γδ T cellinhibitors. Alternatively, by selectively targeting γδ T cellsexpressing Vγ4 (or the human equivalent thereof), the timing of thetreatment may be effective at later timepoints. Other methods fordirecting the method of the present invention to γδ T cells, includingto specific subsets of γδ T cells are discussed below.

Accordingly, the method of the present invention can be carried out byany suitable process of increasing the numbers, activation or biologicalactivity, or survival of γδ T ciells, wherein increased action of γδ Tcells is effective to reduce airway hyperresponsiveness in a mammal.Such a process can be performed in vivo, such as by administration of acompound to an animal which increases the action of γδ T cells in theanimal or by transferring γδ T cells into an animal from another source.Alternatively, such a process can be performed ex vivo, such as byremoving a sample of cells, tissues or bodily fluids from an anysuitable tissue or region in an animal which includes γδ T cells;expanding, activating and/or selecting (isolating) γδ T cells in vitroto increase the number and/or action of γδ T cells in the sample; andreturning at least the γδ T cells to the lung tissue of the animal.

In one embodiment, the method of the present invention includes the useof a variety of agents (i.e., regulatory compounds) which, by acting onγδ T cells, increase the proliferation, activationibiological activity,and/or survival of γδ T cells in the lung tissue of an animal, and/orthe recruitment of other regulatory γδ T cells to the lung tissue of theanimal, such that airway hyperresponsiveness is reduced in the animal.Such agents are generally referred to herein as γδ T cell agonists.According to the present invention, a γδ T cell agonist is any agentwhich increases, typically by direct action on the cell, theproliferation, activationibiological activity, and/or survival of γδ Tcells, and includes agents which act directly on the γδ T cell receptor.A γδ T cell agonist, as referred to herein, can further include, forexample, compounds that are products of rational drug design, naturalproducts, and compounds having partially or fully defined γδ T cellstimulatory properties. A γδ T cell agonist can be a protein-basedcompound, a carbohydrate-based compound, a lipid-based compound, anucleic acid-based compound, a natural organic compound, a syntheticallyderived organic compound, an antibody, or fragments thereof A variety ofknown γδ T cell agonists are described below and all are encompassed bythe present invention.

In one embodiment, γδ T cells are selectively stimulated by randomheterocopolymers of glutamic acid and tyrosine, generally referred toherein as polyGT. polyGT is most commonly known as a randomlysynthesized heterocopolymeric peptide composed of glutamic acid andtyrosine, with an average length of 100 amino acids and a capacity toelicit strong immune responses in certain mouse strains. As described indetail in Cady et al. (2000, J Immunol. 165:1790), polyGT stimulatespolyclonal proliferation of normal (e.g., splenic) γδ T cells as well ashybridomas, but not an cells. Therefore, polyGT is useful forselectively stimulating γδ T cells in the absence of stimulating αβ Tcells, and in the absence of additional targeting of the polyGT to γδ Tcells. According to the present invention, polyGT can be provided as asynthetic peptide, such as polyGlu⁵⁰Tyr⁵⁰ (publicly available fromSigma, P-0151), as a random heterocopolymer of glutamic acid andtyrosine of any other length which is sufficient to elicit a γδ T cellresponse (i.e., stimulation, activation), and by several naturalproteins which contain such repeats, including predicted and actualproteins in bacteria, viruses, mice and humans. Such sequences, and theproteins containing such sequences, can be readily identified byperforming simple sequence searches in the public sequence databases.Such peptides are simple to produce and test for γδ T cell stimulation,using methods formeasuring γδ T cell stimulation as described elsewhereherein and in Cady et al., ibid., which is incorporated herein byreference in its entirety.

In another embodiment, a peptide referred to a polyGAT (1:1:1) is alsostimulatory for γδ T cells and can be used in the present method.PolyGAT is a synthetic peptide that is comprised of glutamic acid,alanine and tyrosine in a 1:1:1 ratio. The peptide was named prior tothe now universally standard single letter code for amino acids.

Yet another γδ T cell agonist includes synthetic AT and otheroligonucleotides (i.e., nucleic acid sequences having from about 5 toabout 100 nucleotides, and more preferably from about 5 to about 50, andmore preferably from about 5 to about 30 nucleotides). Synthetic AT isan oligonucleotide of at least 5 nucleotides composed of adenosine andtbymidine. Other types of oligonucleotides, including those composed ofguanine and cytosine, are also stimulatory for γδ T cells. Shorteroligonucleotides (less than 20 nucleotides) stimulate only whenimmobilized (e.g., on any suitable substrate) or otherwise polymerized.

In one embodiment of the present invention, the agent used forincreasing γδ T cell action is an antibody. In one aspect, the antibodyselectively binds to a γδ T cell in a manner such that the γδ T cellproliferation, survival or activation is increased. In a preferredaspect, the antibody selectively binds to the γδ T cell receptor (γδTCR) and activates the γδ T cell by such binding. In one aspect, theantibody selectively binds to a specific subset of γδ T cell receptorswhich are identified as being particularly effective to reduce airwayhyperresponsiveness in an animal. In a particularly preferredembodiment, the antibody binds to a γδ T cell receptor expressing a Vγ4chain. As used herein, the term “selectively binds to” refers to theability of antibodies of the present invention to preferentially bind tospecified proteins (e.g., a γδ T cell receptor). Binding can be measuredusing a variety of methods standard in the art including enzymeimmunoassays (e.g., ELISA), immunoblot assays, radioimmunoassays, etc.Several antibodies which selectively bind to a γδ T cell receptor areknown in the art and are publicly available. Such antibodies include,but are not limited to: anti-TCR-8 (GL3, GL4 and UC₇₋₁₃D5 (PharMingen,San Diego, Calif.); or 403.A10); anti-mouse TCR-Vγ1 (2.11); anti-TCR-Vγ4(UC₃₋₁₀A6; PharMingen, San Diego, Calif.). Isolated antibodies of thepresent invention can include serum containing such antibodies, orantibodies that have been purified to varying degrees. Antibodies of thepresent invention can be polyclonal or monoclonal, functionalequivalents such as antibody fragments (e.g., Fab fragments or Fab₂fragments) and genetically-engineered antibodies, including single chainantibodies or chimeric antibodies, including bi-specific antibodies thatcan bind to more than one epitope.

Generally, in the production of an antibody, a suitable experimentalanimal, such as a rabbit, hamster, guinea pig or mouse, is exposed to anantigen against which an antibody is desired. Typically, an animal isimmunized with an effective amount of antigen that is injected into theanimal. An effective amount of antigen refers to an amount needed toinduce antibody production by the animal. The animal's immune system isthen allowed to respond over a pre-determined period of time. Theimmunization process can be repeated until the immune system is found tobe producing antibodies to the antigen. In order to obtain polyclonalantibodies specific for the antigen, serum is collected from the animalthat contains the desired antibodies. Such serum is useful as a reagent.Polyclonal antibodies can be further purified from the serum by, forexample, treating the serum with ammonium sulfate. In order to obtainmonoclonal antibodies, the immunized animal is sacrificed and Blymphocytes are recovered from the spleen. The differentiating andproliferating daughter cells of the B lymphocytes are then fused withrmyeloma cells to obtain a population of hybridoma cells capable ofcontinual growth in suitable culture medium. Hybridomas producing adesired antibody are selected by testing the ability of an antibodyproduced by a hybridoma to bind to the antigen. Methods of producingboth polyclonal and monoclonal antibodies of a desired specificity arewell known in the art.

Another agent that is particularly useful for increasing the action ofγδ T cells includes a protein or peptide having a corresponding to aconsensus motif that has been identified as being bound by the molecularchaperone known as BiP. This consensus motif is described in detail inBlond-Elguindi et al., 1993, Cell 75:717-728, incorporated herein byreference in its entirety. More particularly, the molecular chaperone,BiP, is the sole member of the HSP70 family localized in the endoplasmicreticulum. BiP is required for translocation fo newly synthesizedproteins across the ER membrane and for their subsequent folding andassembly in the ER lumen. The role of BiP as chaperone depends on itsability to recognize a wide variety of nascent polypeptides that shareno obvious sequence similarity, while discriminating between properlyfolded and unfolded structures. Blond-Elguindi et al. identified aheptameric consensus motif shared by peptides bound by BiP which can beused to predict and identify BiP-binding sites in natural proteins. Itis the peptides consisting essentially of these BiP-binding sites,including such peptides found in various mycobacteria and bacteria,which, without being bound by theory, the present inventors believe maybe particularly stimulatory for γδ T cells. As set forth inBlond-Elguindi, the BiP binding motif is best set forth asHy(W/X)HyXHyXHy, where Hy is a large hydrophobic amino acid (mostfrequently Trp, Leu or Phe), W is Trp, and X is any amino acid. Thiscore motif is of the size determined previously to fill thepeptide-binding pocket of BiP (Flynn et al., 1991, Science 245:385).Additionally, the following amino acid tendencies at various positionshave been observed: Gln is enriched at positions 4 and 8; Met, Gly andThr are enriched at position 3; Asn, Ser and Tyr are enriched atposition 5; and His, Ile, Pro and Thr are enriched at position 7. Thepresent inventors have found that several peptides having this motif arecapable of stimulating γδ T cell responses. Such peptides include thepeptide identified by SEQ ID NO:1 which has the amino acid sequencedenoted FALQLEL. This sequence is an artificial sequence that thepresent inventors have modified from a mycobacterial HSP-60 protein(from M. leprae) (i.e., FGLQLEL, SEQ ID NO:2), both of which the presentinventors have identified as being stimulatory for γδ T cells. SEQ IDNO: 1 was generated by the present inventors to better stimulate γδ Tcell hybridomas (Fu et al., 1994, J. Imnunol. 152:1578). Additionalpeptides having the BiP binding motif from other organisms, includingother mycobacteria, bacteria, yeast, and mammals (human and mouse) haveproven to be stimulatory for γδ T cells (data not shown herein). Suchpeptides, and proteins comprising such peptides in a form which isaccessible to the γδ T cell receptor, are encompassed by the presentinvention for use in increasing the action of γδ T cells.

According to the present invention, peptides suitable for stimulation ofγδ T cells are at least about seven amino acids in length, and caninclude peptides of at least about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 amino acids. In one embodiment, a stimulatory consistingessentially of a given specified peptide (i.e., a peptide having the BiPpeptide-binding motif) can include up to about 10 additional amino acidson either side of the BiP-binding motif. A protein comprising a suitableyoT cell stimulatory peptide is not restricted in size, but must havethe ability to increase γδ T cell action as described herein.

Yet another agent that is useful for increasing the action of γδ T cellsaccording to the present invention is tumor necrosis factor-α (TNF-α).The present inventors have previously described that TNF-α is aparticularly effective mediator of γδ T cell activation (See Lahn etal., 1998, supra). Specifically, investigating very early T cellactivation in mouse and human models ofbacterial infection, the presentinventors measured early cellular activation of T cells and found that,while both murine αβ and γδ T cells responded polyclonally to systemicbacterial infections and to lipopolysaccharides (LPS), γδ T cellsresponded more strongly to the bacteria and to LPS. The presentinventors then identified tumor necrosis factor α (TNF-α) as themediator of the early differential T cell activation, and ofdifferential proliferative responses. The stronger response of γδ Tcells to TNF-α was correlated with higher expression levels of TNF-Rp75,suggesting that this TNF-R determines the differential T cellreactivity. These data indicated that TNF-α is an early preferentialactivator of γδ T cells, connecting γδ T cell functions with those ofcells that produce this cytokine, including activated innate effectorcells and antigen-stimulated T lymphocytes. Now, the present inventorshave additionally demonstrated that TNF-α negatively modulates (i.e.,reduces or controls) airway hyperresponsiveness by activating γδ Tcells. These results are described in detail in Example 6. Therefore,one embodiment of the present invention comprises increasing the actionof γδ T cells in an animal by administering to the animal TNF-α. In oneembodiment, the TNF-α is administered to the lung tissue of an animal.In a preferred embodiment, the TNF-α is targeted to γδ T cells in vivoor ex vivo by one of the methods of selectively targeting γδ T cells asdescribed elsewhere herein.

Other agents useful for increasing the action of γδ T cells includevarious compounds that can be associated with bacteria and/or viruses.Such compounds include, but are not limited to: glycosylated proteins orpeptides, mycobacterial products, and Listeria cell wall products. It isknown that γδ T cells respond during bacterial and viral infections.Additionally, in two mouse models of infection with the facultativeintracellular bacterium Listeria monocytogenes, depletion of γδ T cellsresulted in prolonged and exacerbated inflammation of the target organs,which underwent extensive tissue destruction (Fu et al., supra;Mombaerts et al., 1993, Nature 365:53; Mukasa et al., 1995, J Immunol.155:2047). Depletion of αβ T cells did not have the same consequences,despite comparable or increased bacterial loads. Similar findings werealso recently reported in a mouse model of lung infection withMycobacterium tuberculosis (D'Souza et al., 1997, J Jmmunol. 158:1217).It has not been resolved whether γδ T cell reactivity in theseinfections is directly dependent on antigen recognition by these cells,or instead is merely driven by the innate and adaptive host responses tothe bacteria, although stimulation of γδ T cells by bacterial componentshas been well documented (Haas et al., 1993, Annu. Rev. Immunol.11:637). Particularly strong γδ T cell responses have been noted afterinfection of mice with certain Gram-negative bacteria, includingEscherichia coli and Salmonella strains (Takad a et al., 1993, J.Immunol. 151:2062; Emoto et al., 1992, J. Exp. Med. 176:363; Mixter etal., 1994, Infect. Immun. 62:4618). Although it is controversial whetherγδ T cells contribute to host protection against these pathogens(Weintraub et al., 1997, Infect. Immun. 65:2306), it has beendemonstrated that γδ T cells can be stimulated by lipopolysaccharides(LPS) (Skeen et al., 1993, J. Exp. Med. 178:971; Reardon et al., 1995,J. Invest. Dermatol. 105:585; Tsuji et al., 1996, Int. Immunol. 8:359).The present inventors have recently found that γδ T cells responded morestrongly to two types of systemic bacterial infection and to LPS thandid ad T cells (Lahn et al., 1998, J. Immunol. 160:5221). Finally, thepresent inventors have also previously demonstrated that γδ T cellhybridomas respond in vitro to mycobacterial proteins, includingportions of the mycobacterial heat shock protein HSP65. Without beingbound by theory, the present inventors believe that the activation of γδT cells by this protein may be related to the presence of a BiP-bindingmotif as discussed above. To the extent that portions (i.e., peptides)of the mycobacterial heat shock proteins, and particularly, portions ofthe mycobacterial HSP-60 family proteins, stimulate γδ T cells andthrough this action reduce AHR, such proteins or the portions thereofare encompassed as useful agents by the present invention.

In another embodiment, γδ T cell agonists can include cardiolipin.Cardiolipin is a phospholipid that selectively stimulates γδ T cellhybridomas but not αβ T cell hybridomas. Most other phospholipids testedby the present inventors have not been stimulatory, althoughphosphatidyl-glycerol is weakly stimulatory and therefore, thisphospholipid, or an improved homologue thereof, may also be useful inthe present invention.

For the activation of γδ T cells, the present invention also includesthe use of “phospho-antigens”. Phospho-antigens are antigens containingphosphate groups such as isoprenylpyrophosphate (IPP) and many othersthat have been characterized by the research groups of Michael Brennerand others (e.g., Tanaka et al., 1995, Nature 375:155-158). Yet anotherγδ T cell stimulatory agent includes carbin alkylamines, including thosethat are present in microbes, edible plants and Tea (Bukowski et al.,1999, Immunity 11:57-65).

In one embodiment, γδ T cell agonists of the present invention includeproducts of drug design, including peptides, oligonucleotides,carbohydrates and/or synthetic organic molecules which regulate theproliferation, activation/biological activity, and/or survival of γδ Tcells. Such an agent can be obtained, for example, from moleculardiversity strategies (a combination of related strategies allowing therapid construction of large, chemically diverse molecule libraries),libraries of natural or synthetic compounds, in particular from chemicalor combinatorial libraries (i.e., libraries of compounds that differ insequence or size but that have the same building blocks) or by rationaldrug design. See for example, Maulik et al., 1997, MolecularBiotechnology: Therapeutic Applications and Strategies, Wiley-Liss,Inc., which is incorporated herein by reference in its entirety.Candidate compounds initially identified by drug design methods can bescreened for γδ T cell stimulatory activity and an ability to reduce AHRby increasing the action of γδ T cells using the methods describedelsewhere herein.

In a molecular diversity strategy, large compound libraries aresynthesized, for example; from peptides, oligonucleotides, carbohydratesand/or synthetic organic molecules, using biological, enzymatic and/orchemical approaches. The critical parameters in developing a moleculardiversity strategy include subunit diversity, molecular size, andlibrary diversity. The general goal of screening such libraries is toutilize sequential application of combinatorial selection to obtainhigh-affinity ligands against a desired target, and then optimize thelead molecules by either random or directed design strategies. Methodsof molecular diversity are described in detail in Maulik, et al.,stipra.

In a rational drug design procedure, the three-dimensional structure ofa regulatory compound can be analyzed by, for example, nuclear magneticresonance (NMR) or X-ray crystallography. This three-dimensionalstructure can then be used to predict structures of potential compounds,such as potential regulatory agents by, for example, computer modeling.The predicted compound structure can be used to optimize lead compoundsderived, for example, by molecular diversity methods. In addition, thepredicted compound structure can be produced by, for example, chemicalsynthesis, recombinant DNA technology, or by isolating a mimetope from anatural source (e.g., plants, animals, bacteria and fungi).

Various other methods of structure-based drug design are disclosed inMaulik et al., 1997, supra. Maulik et al. disclose, for example, methodsof directed design, in which the user directs the process of creatingnovel molecules from a fragment library of appropriately selectedfragments; random design, in which the user uses a genetic or otheralgorithm to randomly mutate fragments and their combinations whilesimultaneously applying a selection criterion to evaluate the fitness ofcandidate ligands; and a grid-based approach in which the usercalculates the interaction energy between three dimensional receptorstructures and small fragment probes, followed by linking together offavorable probe sites.

In one embodiment, additional regulators γδ T cells can be identified byusing high-throughput screening methods, including DNA arrays.

In accordance with the present invention, acceptable protocols toadminister an agent (i.e., an agent/compound that increases γδ T cellactivation), including the route of administration and the effectiveamount of an agent to be administered to an animal, can be determinedand accomplished by those skilled in the art. An agent of the presentinvention can be administered in vivo or ex vivo. Suitable in vivoroutes of administration can include, but are not limited to, oral,nasal, inhaled, topical, intratracheal, transdenial, rectal, andparenteral routes. Preferred parenteral routes can include, but are notlimited to, subcutaneous, intradermal, intravenous, intramuscular, andintraperitoneal routes. Preferred topical routes include inhalation byaerosol (i.e., spraying) or topical surface administration to the skinof a mammal. Most preferably, an agent is administered to the lungtissue of an animal. Routes suitable for delivery of an agent to thelung tissue include, but are not limited to: nasal, inhaled,intratracheal, or intravenous routes. Most preferably, an agent isadministered to an animal by nasal, inhaled, or intratracheal routes.

Ex vivo refers to performing part of the administration step outside ofthe patient, such as by removing cells from a patient, culturing suchcells in vitro to increase γδ T cell action, and returning the cells, ora subset thereof (e.g., isolated γδ T cells) to the patient. Ex vivomethods are particularly useful because the γδ T cells in the lung ofthe patient can be isolated from other cells in vitro, andexpanded/activated prior to return of the cells to the lung of thepatient. Therefore, it is not necessary to specifically isolate γδ Tcells from a patient, but rather, a tissue, cell population and/orbodily fluid containing γδ T cells can be initially isolated, followedby stimulation of the cells by yb-specific or non-specific methods of Tcell stimulation. Either prior to or subsequent to such stimulation, ifdesired, the γδ T cells can be isolated for return to the patient as asubstantially homogeneous γδ T cell population in which γδ T cell actionhas been increased. It is noted, however, that separation of the γδ Tcells from the other cells removed from the patient is not required andin some circumstances, may not be desirable (e.g., other cells removedfrom the patient might be valuable as being positively affected by γδ Tcell activation). In this instance, γδ T cells can be selectivelyactivated and/or expanded ex vivo, and returned to the patient with theother cells.

Preferably, in an ex vivo method, the sample containing the γδ T cellsto be manipulated is obtained from the lung tissue of the patient.Methods for obtaining cells from and returning cells to the lung of ananimal, including bronchoalveolar lavage, are well known in the art. Inaddition, as described above, methods for manipulating γδ T cells exvivo (i.e., iii vitro) are also well known in the art.

According to the method of the present invention, an effective amount ofa agent that increases γδ T cell action (also referred to simply as “anagent”) to administer to an animal comprises an amount that is capableof reducing airway hyperresponsiveness (AHR) without being toxic to themammal. An amount that is toxic to an animal comprises any amount thatcauses damage to the structure or function of an animal (i.e.,poisonous).

In one embodiment, the effectiveness of an agent that increases γδ Tcell action to protect an animal from AHR in an animal having or at riskof developing AHR can be measured in doubling amounts. For example, theability of an animal to be protected from AHR (i.e., experience areduction in or a prevention of) by administration of a given γδ T cellagonist is significant if the animal's PC_(20methacholine)FEV₁ is at 1mg/ml before administration of the γδ T cell agonist and is at 2 mg/mlof Mch after administration of the γδ T cell agonist. Similarly, a γδ Tcell agonist is considered to be effective if the animal'sPC_(20methacholine)FEV₁ is at 2 mg/ml before administration of the γδ Tcell agonist, and is at 4 mg/ml of Mch after administration of the γδ Tcell agonist. Methods for measuring an animal's PC_(20methacholine)FEV₁have been described above and are well known in the art.

In one embodiment of the present invention; in an animal that has AHR,an effective amount of an agent to administer to an animal is an amountthat measurably reduces AHR in the animal as compared to prior toadministration of the agent. In another embodiment, an effective amountof an agent to administer to an animal is an amount that measurablyreduces AHR in the animal as compared to a level of airway AJR in apopulation of animals with inflammation that is associated with AHRwherein the agent was not administered.

In one embodiment of the present invention, an effective amount of anagent to administer to an animal includes an amount that is capable ofdecreasing methacholine responsiveness without being toxic to theanimal. A preferred effective amount of an agent comprises an amountthat is capable of increasing the PC_(20methacholine)FEV₁ of an animaltreated with the an agent by about one doubling concentration towardsthe PC_(20methacholine)FEV₁ of a is normal animal. A normal animalrefers to an animal known not to suffer from or be susceptible toabnormal AHR. A test animal refers to an animal suspected of sufferingfrom or being susceptible to abnormal AHR.

In another embodiment, an effective amount of an agent according to themethod of the present invention, comprises an amount that results in animprovement in an animal's PC_(20methacholine)FEV₁ value such that thePC_(20methacholine)FEV₁ value obtained before administration of the anagent when the animal is provoked with a first concentration ofmethacholine is the same as the PC_(20methacholine)FEV₁ value obtainedafter administration of the an agent when the animal is provoked withdouble the amount of the first concentration of methacholine. Apreferred amount of an agent comprises an amount that results in animprovement in an animal's PC_(20methacholine)FEV₁ value such that thePC_(20methacholine)FEV₁ value obtained before administration of the anagent is between about 0.01 mg/ml to about 8 mg/ml of methacholine isthe same as the PC_(20methacholine)FEV₁ value obtained afteradministration of the an agent is between about 0.02 mg/ml to about 16mg/ml of methacholine.

As previously described herein, the effectiveness of an agent to protectan animal having or susceptible to AHR can be determined by measuringthe percent improvement in FEV₁ and/or the FEV₁/FVC ratio before andafter administration of the agent. In one embodiment, an effectiveamount of an agent comprises an amount that is capable of reducing theairflow limitation of an animal such that the FEV₁/FVC value of theanimal is at least about 80%. In another embodiment, an effective amountof an agent comprises an amount that is capable of reducing the airflowlimitation of an animal such that the FEV₁/FVC value of the animal isimproved by at least about 5%, or at least about 100 cc or PGFRG 10L/min. In another embodiment, an effective amount of an agent comprisesan amount that improves an animal's FEV₁ by at least about 5%, and morepreferably by between about 6% and about 100%, more preferably bybetween about 7% and about 100%, and even more preferably by betweenabout 8% and about 100% (or about 200 ml) of the animal's predictedFEV₁. In another embodiment, an effective amount of an agent comprisesan amount that improves an animal's FEV₁ by at least about 5%, andpreferably, at least about 10%, and even more preferably, at least about25%, and even more preferably, at least about 50%, and even morepreferably, at least about 75%.

It is within the scope of the present invention that a static test canbe performed before or after administration of a provocative agent usedin a stress test. Static tests have been discussed in detail above.

A suitable single dose of an agent that increases γδ T cell action(i.e., a γδ T cell agonist) to administer to an animal is a dose that iscapable of reducing or preventing airway hyperresponsiveness in ananimal when administered one or more times over a suitable time period.In particular, a suitable single dose of an agent comprises a dose thatimproves AHR by a doubling dose of a provoking agent or improves thestatic respiratory function of an animal. A preferred single dose of anagent typically comprises between about 0.01 microgram×kilogram⁻¹ andabout 10 milligram×kilogram⁻¹ body weight of an animal. A more preferredsingle dose of an agent comprises between about 1 microgram×kilogram⁻¹and about 10 milligram×kilogram⁻¹ body weight of an animal. An even morepreferred single dose of an agent comprises between about 5microgram×kilograms' and about 7 milligram×kilogram⁻¹ body weight of ananimal. An even more preferred single dose of an agent comprises betweenabout 10 microgram×kilograms' and about 5 milligram×kilograms' bodyweight of an animal. A particularly preferred single dose of an agentcomprises between about 0.1 milligram×kilograms and about 5milligram×kilograms body weight of an animal, if the an agent isdelivered by aerosol. Another particularly preferred single dose of anagent comprises between about 0.1 microgram×kilograms⁻¹ and about 10microgram×kilogram⁻¹ body weight of an animal, if the agent is deliveredparenterally. These doses particularly apply to the administration ofprotein agents, antibodies, and/or small molecules (i.e., the productsof drug design).

In one embodiment, the agent is administered with a pharmaceuticallyacceptable carrier, which includes pharmaceutically acceptableexcipients and/or delivery vehicles, for administering the agent to apatient (e.g., a chimeric antibody or a liposome delivery vehicle). Asused herein, a pharmaceutically acceptable carrier refers to anysubstance suitable for delivering an agent useful in the method of thepresent invention to a suitable in viivo or ex vivo site. Preferredpharmaceutically acceptable carriers are capable of maintaining an agentof the present invention in a form that, upon arrival of the agent inthe animal and/or at a target γδ T cell, the agent is capable ofinteracting with its target (i.e., the γδ T cell) such that AHR isreduced or prevented. Suitable excipients of the present inventioninclude excipients or formularies that transport or help transport, butdo not specifically target an agent to a cell (also referred to hereinas non-targeting carriers). Examples of pharmaceutically acceptableexcipients include, but are not limited to water, phosphate bufferedsaline, Ringer's solution, dextrose solution, serum-containingsolutions, Hank's solution, other aqueous physiologically balancedsolutions, oils, esters, glycols and dry-powder inhalers. Aqueouscarriers can contain suitable auxiliary substances required toapproximate the physiological conditions of the recipient, for example,by enhancing chemical stability and isotonicity.

Suitable auxiliary substances include, for example, sodium acetate,sodium chloride, sodium lactate, potassium chloride, calcium chloride,and other substances used to produce phosphate buffer, Tris buffer, andbicarbonate buffer. Auxiliary substances can also include preservatives,such as thimerosal,—or o-cresol, formalin and benzol alcohol.Compositions of the present invention can be sterilized by conventionalmethods and/or lyophilized.

One type of pharmaceutically acceptable carrier includes a controlledrelease formulation that is capable of slowly releasing a composition ofthe present invention into an animal. As used herein, a controlledrelease formulation comprises an agent of the present invention in acontrolled release vehicle. Suitable controlled release vehiclesinclude, but are not limited to, biocompatible polymers, other polymericmatrices, capsules, microcapsules, microparticles, bolus preparations,osmotic pumps, diffusion devices, liposomes, lipospheres, andtransdermal delivery systems. Suitable delivery vehicles include, butare not limited to liposomes, viral vectors or other delivery vehicles,including ribozymes. Natural lipid-containing delivery vehicles includecells and cellular membranes. Artificial lipid-containing deliveryvehicles include liposomes and micelles. A delivery vehicle of thepresent invention can be modified to target to a particular site in apatient, thereby targeting and making use of an agent at that site.Suitable modifications include manipulating the chemical formula of thelipid portion of the delivery vehicle and/or introducing into thevehicle a targeting agent (e.g., an antibody) capable of specificallytargeting a delivery vehicle to a preferred site (e.g., a γδ T cell).Other suitable delivery vehicles include gold particles,poly-L-lysine/DNA-molecular conjugates, and artificial chromosomes. Inone embodiment, when the route of delivery is inhaled, a composition oragent of the present invention can be delivered by an inhaler device.

A pharmaceutically acceptable carrier which is capable oftargeting isherein referred to as a “delivery vehicle.” Delivery vehicles of thepresent invention are capable of delivering a formulation, including anagent that increases the action of γδ T cells, to a target site in amammal. A “target site” refers to a site in a mammal to which onedesires to deliver a therapeutic formulation. For example, a target sitecan be any cell which is targeted by direct injection or delivery usingantibodies (e.g., monospecific, chimeric or bispecific antibodies)liposomes, viral vectors or other delivery vehicles, includingribozymes. Examples of delivery vehicles include, but are not limitedto, antibodies, artificial and natural lipid-containing deliveryvehicles, viral vectors, and ribozymes. Natural lipid-containingdelivery vehicles include cells and cellular membranes. Artificiallipid-containing delivery vehicles include liposomes and micelles. Adelivery vehicle of the present invention can be modified to target to aparticular site in a mammal, thereby targeting and making use of anucleic acid molecule at that site. Suitable modifications includemanipulating the chemical formula of the lipid portion of the deliveryvehicle and/or introducing into the vehicle a compound capableofspecifically targeting a delivery vehicle to a preferred site, forexample, a preferred cell type. Specifically, targeting refers tocausing a delivery vehicle to bind to a particular cell by theinteraction of the compound in the vehicle to a molecule on the surfaceof the cell. Suitable targeting compounds include ligands capable ofselectively (i.e., specifically) binding another molecule at aparticular site. Examples of such ligands include Iiantibodies,antigens, receptors and receptor ligands. Manipulating the chemicalformula of the lipid portion of the delivery vehicle can modulate theextracellular or intracellular targeting of the delivery vehicle. Forexample, a chemical can be added to the lipid formula of a liposome thatalters the charge of the lipid bilayer of the liposome so that theliposome fuses with particular cells having particular chargecharacteristics.

In one embodiment, an agent of the present invention is targeted to a γδT cell by using an antibody that selectively binds to a proteinexpressed on the surface of the target γδ T cell. Preferably, theantibody binds to a γδ T cell receptor, with γδ T cell receptorscomprising a Vγ4 chain being particularly preferred. Such an antibodycan include functional antibody equivalents such as antibody fragments(e.g., Fab fragments or Fab₂ fragments) and genetically-engineeredantibodies, including single chain antibodies or chimeric antibodies,including bi-specific antibodies that can bind to mnore than oneepitope. Such targeting antibodies are complexed with an agent thatincreases the action of the γδ T cell that is targeted, and serves todeliver the agent to the γδ T cell. The antibodies can be complexed tothe target by any suitable means, including by complexing with aliposome, or by recombinant or chemical linkage of the agent to theantibody. In one embodiment, the agent is a second antibody or portionthereof that stimulates a γδ T cell and that forms a chimeric orbispecific antibody with the targeting antibody.

The method of the present invention can be used in any animal, andparticularly, in any animal of the Vertebrate class, Mammalia,including, without limitation, primates, rodents, livestock and domesticpets. Preferred mammals to treat using the method of the presentinvention include humans.

Yet another embodiment of the present invention relates to a method toidentify a compound that reduces or prevents airway hyperresponsivenessby increasing the action of γδ T cells. Such a method includes the stepsof: (a) contacting a putative regulatory compound with a γδ T cell; (b)detecting whether the putative regulatory compound increases γδ T cellaction; and, (c) administering the putative regulatory compound to anon-human animal in which airway hyperesponsiveness can be induced andidentifying animals in which airway hyperresponsiveness is reduced orprevented as compared to in the absence of the putative regulatorycompound. Preferably, the γδ T cell is contacted with the regulatorycompound under conditions wherein, in the absence of the putativeregulatory compound, the action of the γδ T cell is not substantiallyincreased (i.e., is at a resting, or baseline, level). A putativeregulatory compound that increases γδ T cell action and that reduces orprevents airway hyperresponsiveness in the non-human animal is indicatedto be a compound for reducing or preventing airway hyperresponsiveness.

In this method, the step (b) of detecting can include, but is notlimited to, a method selected from the group of measurement ofmeasurement proliferation of said γδ T cell, measurement of cytokineproduction by said γδ T cell, measurement of calcium mobilization insaid γδ T cell, measurement of cytokine receptor expression by said γδ Tcell, measurement of CD69 upregulationby said γδ T cell, measurement ofupregulation of CD44 by said γδ T cell, and measurement of cytoskeletalreorganization by said γδ T cell. Such methods are known in the art andare described above.

In an alternate embodiment, such a method can include the steps of: (a)contacting a putative regulatory compound with an isolated γδ T cell anddetermining whether the putative regulatory compound binds to the γδ Tcell (e.g., preferably to the γδ T cell receptor); an optional step (b)of further detecting whether compounds that bind to γδ T cell in (a)increase the action of γδ T cells in an assay for γδ T cell biologicalactivity (e.g., a proliferation or cytokine assay); and (c)administering the putative regulatory compound to a non-human animal inwhich airway hyperresponsiveness can be induced and identifying animalsin which airway hyperresponsiveness is reduced or prevented as comparedto in the absence of the putative regulatory compound.

Yet another alternate embodiment of the method to identify a compoundthat reduces or prevents airway hyperresponsiveness associated withinflammation, includes the steps of: (a) contacting a cell or celllysate which expresses a γδ T cell receptor with a putative regulatorycompound; (b) detecting whether the putative regulatory compoundstimulates a γδ T cell receptor function selected from the group of γδ Tcell receptor expression, γδ T cell ligand binding or γδ T cell receptorbiological activity (e.g., stimulation of proliferation, stimulation ofcytokine production by a γδ T cell); and (c) administering the putativeregulatory compound to a non-human animal in which airwayhyperresponsiveness can be induced, and identifying animals in whichairway hyperresponsiveness is reduced or prevented as compared to in theabsence of the putative regulatory compound. A putative regulatorycompound that inhibits γδ T cell receptor expression, ligand binding orbiological activity and that reduces or prevents airwayhyperresponsiveness in the non-human animal is indicated to be acompound for reducing or preventing hyperresponsiveness associated withinflammation.

In one alternate embodiment, step (a) of contacting comprises contactingthe putative regulatory compound with a cell or cell lysate containing areporter gene operatively associated with a regulatory element of the γδT cell receptor, and step (b) of detecting comprises detecting increasedexpression of the reporter gene product. In another aspect of thisembodiment, step (a) of contacting comprises contacting the putativeregulatory compound with a cell or cell lysate containing transcripts ofthe γδ T cell receptor, and step (b) of detecting comprises detectingtranslational activation of the γδ T cell receptor transcript.

As used herein, the term “putative” refers to compounds having anunknown or previously unappreciated regulatory activity in a particularprocess. As such, the term “identify” is intended to include allcompounds, the usefulness of which as a regulatory compound of γδ T cellaction for the purposes of reducing airway hyperresponsiveness isdetermined by a method of the present invention.

The above described methods, in one aspect, involve contacting cellswith the compound being tested for a sufficient time to allow forinteraction of the putative regulatory compound with the γδ T cell andin one embodiment, with the γδ T cell receptor expressed by the cell.The period of contact with the compound being tested can be varieddepending on the result being measured, and can be determined by one ofskill in the art. For example, for binding assays, a shorter time ofcontact with the compound being tested is typically suitable, than thenactivation is assessed. As used herein, the term “contact period” refersto the time period during which cells are in contact with the compoundbeing tested. The term “incubation period” refers to the entire timeduring which cells are allowed to grow prior to evaluation, and can beinclusive of the contact period. Thus, the incubation period includesall of the contact period and may include a further time period duringwhich the compound being tested is not present but during which growthor cytokine production is continuing (in the case of a cell based assay)prior to scoring. The incubation time for growth of cells can vary butis sufficient to allow for the binding of the γδ T cell receptor and/orincreased action of the γδ T cell. It will be recognized that shorterincubation times are preferable because compounds can be more rapidlyscreened. A preferred incubation time is between about 1 minute to about72 hours.

The above-described methods for identifying a compound of the presentinvention include contacting a γδ T cell or a γδ T cell lysate with acompound being tested for its ability to bind to and/or regulate theaction of the γδ T cell or its receptor, respectively. The conditionsunder which the cell or cell lysate of the present invention iscontacted with a putative regulatory compound, such as by mixing, areany suitable culture or assay conditions and includes an effectivemedium in which the cell can be cultured or in which the cell lysate canbe evaluated in the presence and absence of a putative regulatorycompound. For example, γδ T cells or other suitable cells expressing aγδ T cell receptor (i.e., the test cells) can be grown in liquid culturemedium or grown on solid medium in which the liquid medium or the solidmedium contains the compound to be tested. In addition, as describedabove, the liquid or solid medium contains components necessary for cellgrowth, such as assimilable carbon, nitrogen and micro-nutrients. Cellsof the present invention can be cultured in a variety of containersincluding, but not limited to, tissue culture flasks, test tubes,microtiter dishes, and petri plates. Culturing is carried out at atemperature, pH and carbon dioxide content appropriate for the cell.Such culturing conditions are also within the skill in the art. Cellsare contacted with a putative regulatory compound under conditions whichtake into account the number of cells per container contacted, theconcentration of putative regulatory compound(s) administered to a cell,the incubation time of the putative regulatory compound with the cell,and the concentration of compound administered to a cell. Determinationof effective protocols can be accomplished by those skilled in the artbased on variables such as the size of the container, the volume ofliquid in the container, conditions known to be suitable for the cultureof γδ T cells, and the chemical composition of the putative regulatorycompound (i.e., size, charge etc.) being tested. A preferred amount ofputative regulatory compound(s) comprises between about 1 nM to about 10mM of putative regulatory compound(s) per well of a 96-well plate.

Suitable cells for use with the present invention include any γδ T celland in assays which only require the expression of a γδ T cell receptor,any cell that has been transfected with and expresses a γδ T cellreceptor. γδ T cells can include normal γδ T cells (i.e., native, ornatural isolates), T cell clones (i.e., a natural isolate that has beenclonally selected and expanded), or γδ T cell hybridomas (i.e., naturalisolates that have been fused with a myeloma cell line to produce animmortalized T cell hybrid). In one embodiment, host cells geneticallyengineered to express a functional γδ T cell receptor that responds toactivation by known stimulators of γδ T cells can be used as an endpointin the assay; e.g., as measured by a chemical, physiological,biological, or phenotypic change, induction of a host cell gene or areporter gene, change in cAMP levels, activity of other intracellularsignal transduction molecules, proliferation, differentiation, etc.Cytokine-producing cells for use with the present invention includemammalian, invertebrate, plant, insect, fungal, yeast and bacterialcells. Preferred cells include mammalian, amphibian and yeast cells.Preferred mammalian cells include primate, non-human primate, mouse andrat. In one embodiment, the test cell (host cell) should express afunctional γδ T cell receptor that gives a significant response tostimulation through the γδ T cell receptor, preferably greater than 2,5, or 10-fold induction over background.

As disclosed above, the present methods also make use of non-cell basedassay systems to identify compounds that can regulate AHR. For example,isolated membranes may be used to identify compounds that interact withthe γδ T cell receptor being tested. Membranes can be harvested fromcells expressing γδ T cell receptors by standard techniques and used inan in vitro binding assay. A ¹²⁵I-labeled γδ T cell receptor ligand isbound to the membranes and assayed for specific activity; specificbinding is determined by comparison with binding assays performed in thepresence of excess unlabeled ligand. Membranes are typically incubatedwith labeled ligand in the presence or absence of test compound.Compounds that bind to the receptor and compete with labeled ligand forbinding to the membranes reduced the signal compared to the vehiclecontrolsamples.

Alternatively, soluble γδ T cell receptors may be recombinantlyexpressed and utilized in non-cell based assays to identify compoundsthat bind to γδ T cell receptors. Recombinantly expressed γδ T cellreceptor polypeptides or fusion proteins containing one or moreextracellular domains of a γδ T cell receptor can be used in thenon-cell based screening assays. Alternatively, peptides correspondingto one or more of the cytoplasmic domains of the γδ T cell receptor orfusion proteins containing one or more of the cytoplasmic domains of theγδ T cell receptor can be used in non-cell based assay systems toidentify compounds that bind to the cytoplasmic portion of the γδ T cellreceptor; such compounds may be useful to modulate the signaltransduction pathway of the γδ T cell receptor. In non-cell based assaysthe recombinantly expressed yoT cell receptor is attached to a solidsubstrate such as a test tube, microtitre well or a column, by meanswell known to those in the art. The test compounds are then assayed fortheir ability to bind to the γδ T cell receptor.

As discussed above, in vitro cell based assays may be designed to screenfor compounds that regulate γδ T cell receptor expression at either thetranscriptional or translational level. In one embodiment, DNA encodinga reporter molecule can be linked to a regulatory element of a γδ T cellreceptor gene and used in appropriate intact cells, cell extracts orlysates to identify compounds that modulate γδ T cell receptor geneexpression, respectively. Appropriate cells or cell extracts areprepared from any cell type that normally expresses a γδ T cell receptorgene, thereby ensuring that the cell extracts contain the transcriptionfactors required for in vitro or in vivo transcription. The screen canbe used to identify compounds that modulate the expression of thereporter construct. In such screens, the level of reporter geneexpression is determined in the presence of the test compound andcompared to the level of expression in the absence of the test compound.

To identify compounds that regulate γδ T cell receptor translation,cells or in vitro cell lysates containing γδ T cell receptor transcriptsmay be tested for modulation of γδ T cell receptor mRNA translation. Toassay for inhibitors of translation, test compounds are assayed fortheir ability to modulate the translation of γδ T cell receptor mRNA inin vitro translation extracts. Compounds that increase the level of γδ Tcell receptor expression, either at the transcriptional or translationallevel, may be useful for reduction of AHR.

Finally, a putative regulatory compound of the present invention can beevaluated by administering putative regulatory compounds to a non-humantest animal (and eventually, to a human test subject) and detectingwhether the putative regulatory compound reduces AHR in the test animal.Animal models of disease are invaluable to provide evidence to support ahypothesis or justify human experiments. For example, mice have manyproteins which share greater than 90% homology with corresponding humanproteins. Preferred modes of administration, including dose, route andother aspects of the method are as previously described herein for thetherapeutic methods of the present invention. The test animal can be anysuitable non-human animal, including any test animal described in theart for evaluation of AHR. The test animal can be, for example, anestablished mouse model of AHR, as previously described (see, forexample, Takeda et al., (1997). J. Exp. Med. 186, 449-454; Renz et al.,1992, J. Allergy Clin. Immunol. 89:1127-1138; Larsen et al., 1992, J.Clin. Invest. 89:747-752; and Saloga et al., 1993, J. Clin. Itivest.91:133-141). This non-human model system is an accepted model of airwayhyperresponsiveness associated with allergic inflammation which sharesmany characteristics with human respiratory conditions associated withallergic inflammation, including airway hyperresponsiveness, airwayfibrosis, increased IgE production, and eosinophilia. More specifically,the mouse model is an antigen-driven minrine system that ischaracterized by an immune (IgE) response, a dependence on a Th2-typeresponse, and an eosinophil response, and is a valid model for studyingallergic inflammation of the airways in mammals, and particularly inhumans. The model is characterized by both a marked and evolvinghyperresponsiveness of the airways. Briefly, as an exemplary protocolfor this murine model, mice (typically BALB/c) are immunizedintraperitoneally with ovalbumin (OVA). The mice are then chronicallyexposed (i.e., challenged) for 8 days (i.e., 8 exposures of 30 minuteseach in 8 days) to aerosolized OVA. It should be noted that bothimmunization and subsequent antigen challenge are required to observe aresponse in mice. To characterize the murine model, pulmonary functionmeasurements of airway resistance (RL) and dynamic compliance (CL) andhyperresponsiveness are obtained as described in Example 1 below.

Compounds identified by any of the above-described methods can be usedin a method for the reduction or prevention of AHR as described herein.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention.

EXAMPLES Example 1

The following example demonstrates that airway reactivity is increasedin the absence of γδ T cells.

The following materials and methods are used throughout the examplesherein, as indicated.

Animals. The influence of γδ and αβ cells on airway hyperresponsiveness(AHR) was assessed in a murine model of allergen-induced, Tcell-dependent asthma. For these experiments, BALB/c mice, C57BL/6 mice,TCR-β^(−/−) mice (mice deficient in αβ T cells and back-crossed ontoC57BL/6 genetic background), and TCR-δ^(−/−) mice (mice deficient in γδT cells and back-crossed onto C57BL/6 genetic background) were purchasedfrom The Jackson Laboratory (Bar Harbor, Me.), and cared for at NationalJewish Medical and Research Center (Denver, Colo.) following guidelinesfor immunodeficient animals.

Sensitization and airway challenge. Mice (BALB/c, C5713L/6, TCR-β^(−/−)and TCR-δ^(−/−)) were treated with hamster Ig (sham depletion) or withmonoclonal antibody against TCR-8 or TCR-β and received one of thefollowing treatments: (1) no ovalbumin (OVA) treatment (denoted “NT”);(2) airway exposure to nebulized OVA (1% in saline) alone, usingultrasonic nebulization (particle-size 3-5 μm²) for 20 minutes on threeconsecutive days (denoted “3N”); or (3) sensitization to OVA byintraperitoneal injection of 20 μg of OVA (Grade V; Sigma) emulsified in2.25 mg alum (Alumimuject®; Pierce, Rockford, Ill.) in a total volume of100 μl on days 0 and 14, followed by aerosolized airway challenge withnebulized OVA on days 28, 29 and 30 (denoted “2ip3N”). Determination ofairway responsiveness and inflammation was assessed 48 hours after thelast nebulized OVA exposure for 3N- and 2ip3N-treated mice. For each ofthese treatments and type of mice, groups of four mice were analyzed ineach independent experiment.

Determination ofairway responsiveness. Airway responsiveness wasassessed as a change in airway function after challenge with aerosolizedmethacholine (MCh) through the airways. Anesthetized and tracheostomizedmice were mechanically ventilated, and lung function was assessed as amodification to known procedures. Briefly, a four-way connector wasattached to the tracheotomy tube (stainless steel cannula, 18-gauge,with two ports connected to the inspiratory and expiratory sides of twoventilators). Ventilation was achieved at 160 breaths per minute and atidal volume of 0.15 ml with a positive end-expiratory pressure of 2-4cm H₂O (model 683; Harvard apparatus, South Natwick, Mass.). AerosolizedMCh was administered for 10 breaths at rate of 60 breaths/min inincreasing concentrations (1.56, 3.125, 6.25 and 12.5 mg/ml MCh forBALB/c mice, 6.25, 12.5, 25, 50 and 100 mg/ml MCh for C57BL/6 mice) witha tidal volume of 0.5 ml by the second ventilator (model SN-480-7-3-2T;Shineno Manufacturing, Tokyo, Japan). The chamber containing the mousewas continuous with a 1.0-liter glass bottle filled with copper gauze tostabilize the volume signal for thermal drift. Transpulmonary pressurewas detected by a pressure transducer with one side connected to thefourth port of a four-way connector and the other side connected to asecond port on the plethysmograph. Changes in lung volume were measuredby detecting pressure changes in the plethysmographic chamber through aport in the connecting tube with a pressure transducer and thenreferenced to a second copper-gauze filled 1.0-liter glass bottle. Flowwas measured by digital differentiation of the volume signal. Lungresistance (R_(L)) and dynamic compliance (C) were continuously computed(Labview; National Instruments, Austin, Tex.) by fitting flow, volumeand pressure to an equation of motion. After each aerosol MCh challenge,the data were continuously collected for 1-5 min. and maximum values ofR_(L), and minimum values of C were used to express changes in murineairway function.

Depletion with monoclonal antibody against TCR. Depletion was achievedby injection into the tall vein of 200 μg hamster monoclonal antibodiesagainst TCR-δ (mixture of GL3 (PharMingen) and 403.A10) or TCR-β(H57-597 (PharMingen)). Sham depletion was accomplished with hamster Ig(Jackson Laboratories, Bar harbor, Me.).

Broncho alveolar lavage (BAL) fluid. Immediately after assessment ofAHR, lungs were lavaged through the tracheal tube with Hank's balancedsolution (HB55 1×1 ml using 1 lavage injection with 1 ml of HBSS at 37°C.). The volume of and number of cells in the BAL fluid were assessed(Coillter Counter; Coulter, Hialeah, Fla.). BAL fluid cells were stainedwith Leukostate (Fischer Diagnostics, Pittsburgh, Pa.) on cytosineslides and differentiated by experimenters “blinded” to sample identitycounting at least 200 cells with a light microscope.

Histologic examination. Lungs were inflated through the tracheas andfixed with 10% formaldehyde. The left lung was excised and embedded inparaffin, and tissue sections 5 μm in thickness were affixed to slidesand deparaffinized. Sections were stained with hematoxlin and eosin andthe inflammatory reaction assessed by light microscopy.

Eosinophils and major basic protein staining. A FITC-conjugated rabbitmonoclonal antibody against mouse major basic protein was used to assesseosinophil numbers by immunohistochemistry. Positive events were countedin the submucosatissue around central airways using the IPLab2 software(Signal Analytics, Vienna, Va.) counting four different sections peranimal.

Statistical analysis. All results are expressed as the mean and standarddeviation (s.d.) except where otherwise indicated. Analysis of variancewas used to determine the levels of difference between all groups. Pairsof groups were compared by unpaired two-tailed Student's t-test. Pvalues were considered significant at 0.05.

Results. As previously shown by studies that established the role of αβT cells in the development of AHR (Hamelmann et al., 1996, J. Exp. Med.183:1719-1729; Takeda et al., 1997, J. Exp. Med. 186:449-454), C57BL/6mice that were systemically sensitized to ovalbumin (OVA) and challengedthrough the airways developed AHR to inhaled methacholine (MCh), whereasuntreated mice or those exposed to OVA only through the airways did not(FIG. 1A). FIG. 1A shows the R_(L) changes in normal CS7BL/6 mice after2ip3N treatment (▪), 3N treatment (□) and untreated (⋄). There were nodifferences in baseline responses to saline in any of these groups. Rbaseline values (in cm H₂O/ml per second) were 0.56±0.04 (2ip3N),0.57±0.03 (3N) and 0.53±0.03 (NT) P(0.05, 2ip3N compared with 3N).

Mice genetically deficient to γδ T cells (T-cell receptor (TCR)-δ^(−/−))also developed AHR, in contrast to mice deficient in αβ T cells(TCR-β^(−/−)) (FIG. 1B). FIG. 1B shows R_(L) changes in TCR-δ^(−/−) (●),TCR-β^(−/−) (▴) and normal C57BL/6 (▪) mice after 2ip3N treatment inset.There were no significant differences indicated in baseline responses tosaline (data not shown). However, in contrast to a report emphasizingthe enhancement of allergic airway inflammation by γδ T cells on AHR(Zuany-Amorim et al., 1998, Science 280:1265-1267), the presentinventors detected increased responsiveness to MCh in the absence of γδT cells, indicating a suppressive effect of γδ T cells on AHR in thismodel. Since both TCR-β^(−/−) And TCR-δ^(−/−) mice had baseline valuesin airway responsiveness similar to those of the genetically normalcontrol mice (data not shown), background airway tone variations couldbe ruled out as an explanation of these differences.

The broncho alveolar lavage (BAL) fluid and lung sections of C57BL/6 andTCR-β^(−/−) mice challenged systemically and through the airways hadsimilar inflammatory infiltrates with increased eosinophil numbers,whereas TCR-β^(−/−) mice lacked such inflammatory infiltrates (FIG. 1D).FIG. 1D shows the BAL fluid cell composition for total cells, eosinophiland macrophages in 2ip3N-treated C57BL/6, TCR-δ^(−/−) and TCR-β^(−/−)mice. Each bar represents data from at least three independentexperiments using 9-12 mice (P<0.05; brackets indicate cell counts beingcompared; histology not shown). Consistent with previous findings,TCR-β^(−/−) mice had lower numbers of eosinophils in the BAL fluid aswell as fewer eosinophils in lung tissue sections, indicating that γδ Tcells also influence the influx of eosinophils to the inflammatorysites.

To exclude the possibility of developmental compensatory mechanisms inthe genetically TCR-deficient mice, γδ T cells were also depleted inTCR-sufficient mice (i.e., wild type mice) by injecting these mice withmonoclonal antibodies against TCR-δ. The results were similar to thosein TCR-δ^(−\−) mice, in that AHR was increased in mice depleted of γδ Tcells. Moreover, no differences after treatment with monoclonalantibodies against TCR-δ were found in the responses between C57BL/6 andBALB/c mice, two strains known to differ in their airway responsivenessafter OVA sensitization and challenge (FIG. 1C). FIG. 1C shows RLchanges in TCR-δ-depleted (▾) or sham-depleted (∇) BALB/c mice after2ip3N treatment. There were no significant differences in baselineresponses to saline in any of these groups. R_(L) baseline values (in cmH₂O/ml per second) were 0.60±0.03 (sham-depleted) and 0.59±0.02(TCR-6-depleted). Each curve represents data from at least threeindependent experiments using 9-12 mice (P<0.05). Thus, theregulatoryeffects of γδ T cells in OVA-induced AHR seem to be independent of thesegenetic differences. The cellular composition in BAL fluid and lungtissue of the antibody-depleted mice was also similar to that found inthe genetically deficient mice (data not shown).

Based on these findings, it was concluded that, during allergic ax Tcell-dependent AHR, γδ T-cell deficiency results in increased airwayresponsiveness, despite a concurrent reduction in eosinophilicinflammation.

Example 2

The following example demonstrates that the effect of γδ T cells onairway hyperresponsiveness does not require systemic sensitization.

Since γδ T-cell deficiency was shown to influence AHR inallergen-sensitized and challenged mice (Example 1), it was nextdetermined whether systemic sensitization with antigen was necessary forthese effects to be shown. In this experiment, the effect γδ T cells onmice sensitize only through the airways was investigated, using theprotocol described above (3N treatment).

Results are shown in FIGS. 2A-21. FIGS. 2A, 2C and 2G illustrate changesin airway resistance (R_(L)); FIGS. 2B, 2D and 2F illustrate changes indynamic compliance (C). FIGS. 2A and 2B show the effects of 3N treatmentin C57BL/6 (□), TCR-δ^(−/−) (●) and TCR-β^(−/−) (Δ) mice. FIGS. 2C and2D show the effects of 3N treatment in sham-depleted (□) and γδ Tcell-depleted (▪) C57BL/6 mice. FIGS. 2E and 2F show the effects of 3Ntreatment in sham-depleted (⋄) and γδ T cell-depleted (♦) BALB/c mice.There were no significant differences in responses to saline in any ofthese groups. RL baseline values in cm-H₂O/ml per second) were 0.59±0.08(TCR-δ^(−/−)); 0.57±0.03 (C57BL/6); 0.59±0.07 (TCR^(−/−)); 0.57±0.03(sham-depleted C57BL/6); 0.62±0.08 (TCR-8-depleted C57BL/6); 0.54±0.04(sham-depleted BALB/c); 0.56±0.04 (TCR-δ-depleted BALB/c). Each curverepresents data from at least three independent experiments using 9-12mice (P<0.05).

FIG. 2G shows the BAL fluid cell composition for total cells (TC),eosinophil (EOS) and macrophages (Mac) in 3N-tested mice; C57BL/6,TCR-δ^(−/−), TCR-β^(−/−) mice. FIG. 2H shows the BAL fluid cellcomposition of these cells in sham-depleted and γδ T cell-depletedC57BL/6 mice. FIG. 2I shows the BAL fluid cell composition for thesecells in BALB/c mice. Each bar represents data from at least threeindependent experiments using 9-12 mice (P<0.05). The results in FIGS.2A-21 demonstrated that TCR-δ^(−/−) mice had a higher level of to airwayresponsiveness than C57BL/6 mice, even when they were exposed to OVAonly through the airways (nebulized OVA on 3 consecutive days; 3Ntreatment). The higher increases in airway responsiveness in TCR-δ^(−/−)mice involved both the larger airways as assessed by airway resistance(RL) (FIG. 2A) and the smaller airways, as demonstrated by changes indynamic lung compliance (FIG. 2B). As expected, no AER was detectable inTCR-67 ^(−/−) mice. In mice treated with antibodies to deplete γδ Tcells changes in airway function again resembled those of thegenetically deficient mice (FIGS. 2C-F). However, despite these obviouschanges in airway function, mice deficient in γδ T cells did notdemonstrate increases when compared to TCR-sufficient control ininflammatory infiltrates in the BAL fluid or the lung tissue (FIGS.2G-2I; histology not shown). Thus, in the absence of systemic antigensensitization and the associated inflammatory response, γδ T celldeficiency was still associated with increased airway responsiveness,indicating a mechanism independent of antigen-specific reactivity, andthus perhaps of αβ T-cell responses.

Example 3

The following example demonstrates that γδ T-cell regulation of AHR isindependent of αβ T cells.

To further elucidate the mechanism of the observed increase in AHRcorresponding to γδ deficiency, the effect of γδ T-cell depletion onairway responsiveness in 3N-treated mice (see Example 1) was assessed inmice genetically deficient in αβ T cells, by injecting TCR-β^(−/−) micewith antibodies agai-β^(−/−) TCR-8 as described in Example 1, followedby evaluation of AHR as described in Example 1. The results of thisexperiment are shown in FIGS. 3A and 3B (sham-depleted (◯) and γδ Tcell-depleted (⊙) CR-β^(−/−) mice).

The reciprocal condition of αβ T-cell depletion in mice geneticallydeficient in γδ T cells was also assessed using an antibody againstTCR-p in TCR-δ^(−/−) mice, results shown in FIGS. 3C and 3D(sham-depleted (LI) and αβ T cell-depleted (E) TCR-β^(−/−) mice). As afurther control, T cell-deficient mice were treated with antibodiesspecific for the type of T cells they were lacking (non-relevanttreatments). Changes in airway resistance (RL) are shown in FIGS. 3A and3C; changes in dynamic compliance (Cdy.) are shown in FIGS. 3B and 3D.There were no significant differences in baseline responses to saline inany of these groups in FIGS. 3A-3D). RL baseline values (in cm H₂O/mlper second) were 0.59±0.08 (sham-depleted TCR-β^(−/−)); 0.58±0.03(TCR.8-depleted TCR-β^(−/−)); 0.59±0.07 (sham-depleted TCR-δ^(−/−));0.57±0.02 (TCR-3-depleted TCR-δ^(−/−)). Sham-depleted mice did not reactto nebulized saline exposure alone (open triangles, FIGS. 3A and 3B;open diamonds, FIGS. 3C and 3D). Each curve represents data from atleast three independent experiments using 9-12 mice (P<0.05).

FIGS. 3E and 3F illustrate BAL fluid cell composition for total cells(TC) eosinophil (EOS) and macrophages (Mac) in 3N-treated mice;sham-depleted and γδ T cell-depleted TCR-β^(−/−) mice (FIG. 3E); and insham-depleted and αβ T cell-depleted TCR-δ^(−/−) mice (FIG. 3F). Eachbar represents data from at least three independent experiments using9-12 mice.

FIGS. 3A-3D demonstrate that only the depletion of γδ T cells inTCR-β^(−\−) mice resulted in increases in AHR. Depletion of αβ T cellsin TCR-δ^(−/−) mice caused a small decrease in AHR. The non-relevanttreatments did not produce substantial effects (not shown). Again, BALfluid (FIGS. 3E and 3F) and lung tissue (not shown) had no inflammatoryinfiltrates with eosinophil in any of these mice.

Example 4

The following example demonstrates that γδ T-cell regulation isindependent of B-cell and cytokine responses.

Since ovalbumin (OVA)-specific immunoglobulin (Ig) production orT-helper 2 (Th2) associated cytokines have been implicated in thedevelopment of AHR, serum OVA-specific immunoglobulin production(including IgG₁, IgG₂a, and IgE) as well as interleukin (IL)-4, IL-5 andgamma interferon (IFN γ) levels were measured in the BAL fluid ofC57B.L/6, TCR-β^(−/−) and TCR-δ^(−/−) mice after 3N (See Example 1:airway exposure to nebulized OVA alone) or 2ip3N treatment (See Example1: systemic sensitization to OVA).

Measurement ofaantibody against OVA. Serum levels of OVA-specific IgG₁,IgG_(2a), and IgE were measured by ELISA. Briefly, serum samples wereadded to Immulon 2 plates (Dynatech, Chantilly, Virginia) coated with 5μg/ml OVA. OVA-specific IgE was detected with biotinylated antibodyagainst IgE (PharMingen, San Diego, Calif.) and amplified by anavidiniorseradish-peroxidate (Sigma). OVA-specific IgG₁ and IgG₂ weredetected with alkaline phosphatase labeled antibodies (PharMingen, SanDiego, Calif.). OVA-specific antibody titers of samples were related toan internal “pooled” standard arbitrarily assigned to be 100 ELISA units(EU). FIGS. 4A-4C show individual levels of OVA-specific IgG₁, (FIG.4A), IgE (FIG. 4B) and IgG_(2a) (FIG. 4C), as defined by ELISA units toan OVA standard, for mice (horizontal axis, mouse strain, n=7-12) leftuntreated (◯) or given 3N(●) or 2ip3N (▪) treatment. Crosses representthe means of the immunoglobulin levels (horizontal axis, mean±s.e.m.;P<0.05) between levels of immunoglobulin of 3N and 2ip3N-treated mice.

As shown in FIGS. 4A-4C, after 3N treatment, no significant OVA-specificIg levels were detected in any of the mice, including TCR-δ^(−/−) mice,despite the fact that the TCR-δ^(−/−) mice showed increased airwayresponsiveness after this treatment. As expected, after 2ip3N treatment,αβ T cell-sufficient mice showed increased levels of OVA-specific Igproduction whereas αβ T cell-deficient mice did not. OVA-specific IgGmay be an exception to this observation, because some of the TCR-β^(−\−)mice showed increased levels after 2ip3N treatment (FIG. 4C).

Cytokine levels in BALfluid. The levels of IFN-γ, IL-4 and IL-5 in BALfluid were assessed by ELISA. Briefly, samples were added to Immulon 2plates (Dynatech, Chantilly, Va.) coated with monoclonal antibodiesagainst IFN-γ (clone R4-6A2), IL-4 (clone 11B11) or IL-5 (clone TRFK-5)(all from PharMingen, San Diego, Calif.). Biotinylated monoclonalantibodies against IFN-γ (clone XMG 1.2), IL-4 (clone BVD6-24G2) or IL-5(clone TRFK-4) (all from PharMingen, San Diego, Calif.) were used foramplified detection. Cytokine levels were calculated by comparison withknown cytokine standards with a detection limit of 4 pg/ml for eachcytokine. FIGS. 5A-5C show the concentration (pg/ml) of IL-5 (FIG. 5A),IL-4(FIG. 5B) and IFN-γ (FIG. 5C) in BAL fluid of mice (horizontal axis,strains; n=7-12 mice per treatment) after receiving no treatment (□), 3N(▪) treatment, or 2ip3N (▪) treatment (error bars: s.e.m.). There wereno statistical differences between 3N-treated and 2ip3N-treated mice.

None of the cytokines assessed in the BAL fluid were increased inTCR-δ^(−/−) mice after 3N treatment (FIGS. 5A-5C), despite increasedairway responsiveness after this treatment. The same cytokines wereincreased after 2ip3N treatment in the αβ T cell-sufficient mice, butnot in TCR-β^(−/−) mice. However, TCR-β^(−/−) mice had increasedbaseline levels for all of the tested cytokines, despite their lack ofairway responsiveness after either 3N or 2ip3N treatments.

The results described here demonstrate a previously unknown γδ Tcell-dependent mechanism in the regulation of airway responsiveness,which is independent of αβ T cells and their allergen-specificresponses. This experiment shows no evidence to indicate that antibodiesare involved in this regulatory mechanism. Furthermore, the increasedairway responsiveness in TCR-δ^(−/−) mice was not correlated withincreases in cytokine levels previously suggested to be involved inmodels allergic inflammation (McMenamin et al., 1994, Science265:186-1871; Zuany-Amorim et al., 1998, supra). These results differfrom earlier reports, which have emphasized the role of γδ T cells inregulating allergic αβ T-cell and allergen specific B-cell responses, ortheir role in promoting allergen-induced eosinophilia and IgE responses(McMenamin et al., 1994, supra; Zuany-Amorim et al., 1998, supra;Schramm et al., 1999, International Conference of the American ThoracicSociety; vol. 159:A255 (American Journal of Respiratory and CriticalCare Medicine, San Diego, Calif.)). The mechanism of γδ T cell-dependentregulation of airway responses described herein is therefore notrestricted to allergic inflammation.

Example 5

The following example demonstrates that, in addition to beingindependent of αβ T cells, the γδ T cell-dependent regulatory effects onairway responsiveness described herein are not connected to γδ Tcell-dependent eosinophilia, further emphasizing the differences frompreviously reported γδ T cell-dependent mechanisms.

In addition to the independence of αβ T cells, the γδ T cell-dependentregulatory effects on airway responsiveness described herein are incontrast to γδ T cell-dependent eosinophilia, further emphasizing thedifferences from previously reported γδ T cell-dependent mechanisms(McMenamin et al., 1994, supra; Zuany-Amorim et al., 1998, supra). Tocompare the results described herein with earlier studies, the moreextensive systemic sensitization protocol of previous studies, involvingseven intraperitoneal inj ections of OVA over 14 days, was evaluated.Using these conditions, differences in AHR between wild-type and γδ Tcell-deficient mice were no longer observed, in agreement with theearlier studies (data not shown). The previous findings of γδ Tcell-dependent lung eosinophilic infiltrates was also confirmed (datanot shown). Thus, extensive systemic sensitization seems to promoteimmune-dependent facets of γδ T-cell functions, including γδ Tcell-dependent eosinophilia. Whether or not γδ T cells arem actuallycapable of recognizing OVA remains undetermined. The present findings inthe TCR-β^(−\−) mice sensitized and challenged with OVA do notspecifically address allergen-specific activation of γδ T cells in thedevelopment of AHR or eosinophilic inflammation. In TCR-γδ^(−/−) micedepleted with monoclonal antibody against TCR-β and exposed to airwayaerosolized OVA alone, airway responsiveness was slightly diminished(FIGS. 3C and 3D). Therefore, it remains possible that three exposuresto aerosolized OVA alone activate αβ T cells, especially when thenegative regulation by γδ T cells is absent.

Because the regulatory effects on AHR in the conditions of the presentstudy were associated with a reduction in eosinophil infiltration,eosinophil-independent mechanisms must be considered, althougheosinophil activation itself was not monitored. The cell entities thatγδ T cells could influence include alveolar macrophages, airwayepithelial cells and airway smooth muscle cells. γδ T cells have alreadybeen implicated in regulatory effects involving alveolar macrophages intuberculosis. γδ T cells can alter the development of alveolarmacrophage populations, as untreated TCR-δ^(−/−) mice have lowermacrophage cell counts in BAL fluid than their T cell-sufficient controlcounterparts (data not shown). This action could relate to the findingthat TCR-δ^(−/−) mice are deficient in monocyte chemoattractant protein1 (DiTirro et al., 1998, Infec. Immun. 66:2284-2289). Airway epithelialcells are another source of reactive mediators leading to AHR (King etal., 1999, J. Imunol. 162:5033-5036). The intraepithelial/submucosalocalization of γδ T cells facilitates their reaction to epithelial cellchanges. As do other intraepithelial γδ T cells, lung γδ T cells mayprovide mediators for epithelial repair processes and other epithelialresponses elicited by AHR-inducing stimuli. Furthermore,intraepithelial/submucosa ybT cells could exert their regulatory effectsdirectly on airway smooth muscle cells, for example, by modifyingsecretin of smooth muscle cell derived cytokines, such as GM-CSF, IL-5and IL-4.

In summary, these data demonstrate a previously unknown, αβ Tcell-independent and probably also B cell-independent mechanism ofairway regulation by γδ T cells. This mechanism may co-exist withimmunoregulatory effects of γδ T cells on αβ T cell-dependent pathwaysof AHR.

Example 6

The following example demonstrates that airway hyperresponsiveness isincreased in the absence of tumor necrosis factor-α (TNF-α), and that γδT cells play a role in the failure of TNF-α transgenic mice to developairway hyperresponsiveness.

Airway Hyperresponsiveness is Increased in the Absence of TNF-α

First, airway responsiveness to inhaled Mch was assessed in TNF-αdeficient mice. Female C57BL/6 mice from 8 to 10 weeks of age werepurchased from the Jackson Laboratories (Bar Harbor, Me.). Micegenetically deficient for TNF-α were a gift from Dr. John Harty,University of Iowa, Iowa City, Iowa. These mice were originally derivedfrom intercrosses of (129Sv×C57BL/6)F1 mice heterozygous for the mutated129/Sv TNF-a gene and maintained as a line of mixed 129/B6 geneticbackground homozygous for the mutation since 1996. The mice weremaintained on OVA-free diets. All experimental animals used in thisstudy were under a protocol approved by the Institutional Animal Careand Use Committee of the National Jewish Medical and Research Center.

Both OVA-sensitized and non-sensitized TNF-α deficient mice werechallenged with an aerosol of OVA on three consecutive days, in parallelwith C57BL/6 controls. Briefly, each strain of mouse was grouped basedon the following treatments (4 mice/group/experiment): (a) airwaychallenge (×3) with OVA nebulization alone (N group); or (b)intraperitoneal sensitization with OVA and OVA airway challenge (IPNgroup). Mice were sensitized by intraperitoneal injection of 20 μg ofOVA (Grade V; Sigma) emulsified in 2.25 mg alum (AlumImuject; Pierce,Rockford, Ill.) in a total volume of 100 μl on days 0 and 14. Mice werechallenged via the airways to OVA (1% in saline) for 20 min. on days 28,29 and 30 by ultrasonic nebulization (De Vilbiss, particle size 1-5 μm).Lung resistance (R_(L)) and dynamic compliance (Cdyn) were assessed 48hrs after the last allergen challenge, and the mice were sacrificed toobtain tissues and cells for further assays. Airway resistance and Cdynwere determined as described above in Example 1.

Following OVA sensitization and challenge, C57BL/6 mice developedsignificant increases in RL and decreases in Cdyn in a dose-dependentmanner, compared to mice only challenged with OVA (data not shown). Micegenetically deficient in TNF-α developed AHR and to a greater extentthan the C57BL/6 animals. In non-sensitized mice receiving airwaychallenge alone, the degree of responsiveness was only slightly higherin the TNF-α deficient mice.

The number and types of inflammatory cells in the airways of TNF-αsufficient and deficient mice were measured in bronchoalveolar lavagefluid (BALF) (data not shown). Briefly, after assessment of R_(L) andCdyn, lungs were lavaged via the tracheal tube with Hank's balanced saltsolution, (HBSS, 1×1 ml, 37° C.). The volume of collected BALF wasmeasured in each sample and the number of BALF cells was counted by cellcounter (Coulter Counter; Coulter Co., Hialeah, Fla.). Cytospin slideswere stained with Leukostat (Fisher Diagnostics, Pittsburgh, Pa.) anddifferentiated in a blinded fashion by counting at least 300 cells underlight microscopy.

Cytokine levels (IL-4, IL-5, IL-10, and IFN-γ) in BALF supernatants weremeasured by ELISA as described in Example 4 above. Cytokine levels weredetermined by comparison with the known standards. The limits ofdetection were 4 pg/ml. In C57BL/6 mice, sensitization and challenge toOVA resulted in a marked increase in inflammatory cell numbers comparedwith challenge alone. TNF-α deficient mice showed a similar inflammatorycell response, but the numbers of eosinophils in BALF were significantlylower than in C57BL/6 mice (data not shown).

Inflammatory cells were also measured in the peribronchial andperivascular tissue.

Briefly, lung cells were isolated as previously described (28) andpassed through nylon wool columns to yield an enriched T cellpreparation containing >90% CD3+cells. For cytofluorographic analysis,mAbs were conjugated with N-hydroxysuccinimido-biotin (Sigma) and/orfluorescein isothiocyanate isomer I on Celite (Sigma). Then, 1-2×10⁶cells in 96-well plates (Falcon-Becton Dickinson, Franklin Lakes, N.J.)were stained by using one- or two-color techniques and analyzedcytofluorographically on XL2 (Coulter, Miami, Fla.) counting 150,000events per gated region. For each of the gated populations, meanfluorescence intensity (MFI) was examined to assess shifts influorescence of the examined populations. Streptavidin-phycoerythrin(diluted at 1:100 per 1×10⁶ cells, Tago Immunologicals Biosource,Camarillo, Calif.) was used for the biotin-conjugated antibodies toenhance detection.

In mice challenged only, very little inflammatory cell infiltration wasdetected whereas intraperitoneal sensitization and subsequent challengewith OVA via the airways increased the number of eosinophils andlymphocytes at these sites. Inflammatory cell infiltration insensitized/challenged TNF-α deficient mice was similar to that insensitized and challenged C57BL/6 animals (data not shown).

γδ T Cells in SP-C-TNF-α Transgenic Mice.

For the following experiments, mice expressing the TNF-a gene under thecontrol of the surfactant SP-C promotor (SP-C-TNF-α transgenic mice)were a gift from Dr. Yoshitaka Miyazaki, Department of ClinicalImmunology, Medical Institute of Bioregulation, Kyushu University,Beppu, Japan. The transgenic founder mice (C57BL/6×DBA/2 F1) werebackcrossed with C57BL/6 mice to generate F1 hybrid transgenic mice andmaintained as a heterozygous line by repeated backcrossing since 1995.All transgenic mice were identified by PCR analysis of genomic DNA.Littermate transgene-negative mice were used as controls.

An increased frequency of γδ T cells has been demonstrated in theSP-C-TNF-α transgenic mice (Nakama et al., Exp. Lung Res., 24:57-70,1998). The present inventors therefore investigated the effects of TCR-δmAb on the γδ T cell populations in the lung in OVA sensitized andchallenged TNF-α deficient and transgenic mice. Briefly, depletion wasachieved following injection of 200 μg hamster IgG mAb anti-TCR-8 (1:1mixture of GL3 and 403A10) into the tail vein 3 days prior to the firstOVA challenge. Sham-depletion was carried out using hamster IgG (JacksonLaboratories, Bar Harbor, Me.). OVA sensitization and challenge wascarried out as described above. γδ T cells in the lung were analyzed byflow cytometric analysis.

The number of γδ T cells in the lung in TNF-α deficient mice wassignificantly lower than in normal C57BL/6 mice (data not shown). Incontrast, the number of γδ T cells in the transgenic mice wassignificantly increased compared to littermate transgene-negative mice.Injection of TCR-δ mAb significantly suppressed the numbers of γδ Tcells in the lung in sensitized and challenged transgenic mice as wellas in C57BL/6 and littermate transgene-negative mice; the lower numbersin the TNF-α deficient mice did not change significantly (data notshown).

Airway Responsiveness in TNF-α Transgenic Mice Following γδ T cellDepletion.

As described in Examples 1-6, the present inventors demonstrated that γδcells play a role in the regulation of airway responsiveness (Examples1-6 and Lahn et al., Nature Med., 5:150-1156, 1999). In view of theincreased number of γδ T cells in the TNF-α transgenic mice (Nakama etal., Exp. Lung Res., 24:57-70, 1998) and the findings that γδ T cellsare activated by TNF-α (more so than aγδ T cells) (Lahn et al., J.Immunol., 160:5221-5230, 1998), the present inventors examined whetheractivated γδ T cells might play a role in the failure of TNF-αtransgenic mice to develop AHR. To deplete γδ T cells, TNF-α transgenicmice and TNF-α deficient mice were treated with TCR-8 mAb 3 days beforethe first challenge. TNF-α deficient mice administered anti-TCR-δ failedto show any further increase in AHR (data not shown). In contrast,SP-C-TNF-α transgenic mice depleted of γδ T cells developed AHR whilesham-treated controls did not (data not shown). This effect on AHR wasnot correlated with a cellular inflammatory response: in both OVAsensitized and challenged TNF-α deficient and transgenic mice, therewere no significant differences in the composition of inflarmnatorycells in the BALF following depletion of γδ T cells (data not shown).

In summary, these data confirm that γδ T cells play an important role inthe pathophysiology of the development of AHR and, based on the data inthe SP-C-TNF-α transgenic mice, a possibility as to the mechanism issuggested. Thus, the interactions between TNF-α and γδ T cells may becentral in regulating airway tone following airway exposure to allergen.These findings emphasize complex but important contributions of TNF-α tothe overall regulation of allergic inflammatory responses in the lungand the development of altered airway function in part throughinteractionswith γδ T cells. The additional finding that the absence ofTNF-α was associated with increased levels of IL-1O, an important factorin the development of AHR, reveals another potential mechanism by whichTNF-α may control airway responsiveness, that is through suppression ofIL-10.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

1. A method to reduce airway hyperresponsiveness in a mammal, comprisingincreasing γδ T cell action in a mammal that has, or is at risk ofdeveloping, a respiratory condition associated with airwayhyperresponsiveness.
 2. The method of claim 1, wherein said step ofincreasing γδ T cell action comprises increasing the number of γδ Tcells in the lung tissue of said mammal.
 3. The method of claim 2,wherein said step of increasing comprises removing γδ T cells from saidmammal, inducing said γδ T cells to proliferate ex vivo to increase thenumber of said γδ T cells, and returning said γδ T cells to the lungtissue of said mammal.
 4. The method of claim 1, wherein said step ofincreasing γδ T cell action comprises activating γδ T cells in saidmammal.
 5. The method of claim 4, wherein said step of activating γδ Tcells is performed ex vivo.
 6. The method of claim 1, wherein said stepof increasing γδ T cell action comprises administering an agent to saidmammal that activates γδ T cells in said mammal. 7-16. (Cancelled) 17.The method of claim 6, wherein said agent is targeted to γδ T cells insaid mammal.
 18. The method of claim 17, wherein said agent is targetedto γδ T cells in the lung tissue of said mammal.
 19. The method of claim17, wherein said agent is targeted to γδ T cells having a T cellreceptor (TCR) selected from the group consisting of a murine TCRcomprising Vγ4 and a human TCR comprising Vγ1. 20-21. (Cancelled) 22.The method of claim 6, wherein said agent is administered to the lungtissue of said mammal.
 23. The method of claim 22, wherein said agent isadministered by a route selected from the group consisting of inhaled,intratracheal and nasal routes.
 24. The method of claim 6, wherein saidagent is administered to said animal in an amount effective to reduceairway hyperresponsiveness in said animal as compared to prior toadministration of said agent.
 25. The method of claim 6, wherein saidagent is administered with a pharmaceutically acceptable excipient. 26.The method of claim 1, wherein said γδ T cell action is increased withinbetween about 1 hour and 6 days of an initial diagnosis of airwayhyperresponsiveness in said mammal.
 27. The method of claim 1, whereinsaid γδ T cell action is increased within less than about 72 hours of aninitial diagnosis of airwayhyperresponsiveness in said mammal.
 28. Themethod of claim 1, wherein said γδ T cell action is increased prior todevelopment of airway hyperresponsiveness in said mammal.
 29. The methodof claim 1, wherein said step of increasing γδ T cell action decreasesairway methacholine responsiveness in said mammal. 30-32. (Cancelled)33. The method of claim 1, wherein said airway hyperresponsiveness isassociated with a disease selected from the group consisting of chronicobstructive disease of the airways and asthma.
 34. A method to identifya compound that reduces or prevents airway hyperresponsivenessassociated with inflammation, comprising: a) contacting a putativeregulatory compound with a γδ T cell; b) detecting whether said putativeregulatory compound increases the action of said γδ T cell; c)administering said putative regulatory compound to a non-human animal inwhich airway hyperresponsiveness can be induced, and identifying animalsin which airway hyperresponsiveness is reduced or prevented as comparedto in the absence of said putative regulatory compound; wherein aputative regulatory compound that increases γδ T cell action and thatreduces or prevents airway hyperresponsiveness in said non-human animalis indicated to be a compound for reducing or preventinghyperresponsiveness.
 35. The method of claim 34, wherein said step (b)of detecting is selected from the group consisting of measurementproliferation of said γδ T cell, measurement ofcytokine production bysaid γδ T cell, measurement ofcalcium mobilization in said γδ T cell,measurement of cytokine receptor expression by said γδ T cell,measurement of CD69 upregulation by said γδ T cell, measurement ofupregulation of CD44 by said γδ T cell, and measurement of cytoskeletalreorganization by said γδ T cell.